II SIMPÓSIO DE AVICULTURA DO NORDESTE

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1 AMINO ACID CONSIDERATIONS FOR MODERN BROILERS Introduction Paul B. Tillman, Ph.D.¹, William A. Dozier, III, Ph.D.² ¹Poultry Technical Nutrition Services LLC 5813 Bayside Court Buford, GA ² Professor Department of Poultry Science Auburn University Auburn, AL Digestible amino acids and ratios to lysine are used extensively in formulating diets for poultry (broilers, layers, turkeys, ducks, quail, etc.) on a global basis. Amino acid use has and will continue to increase in the coming decades, especially as high priced ingredients, more limited resources and the environmental impact of excessive dietary nitrogen come more into consideration through life-cycle assessments (McGill et al., 2012, Kidd et al., 2013). The digestible lysine (dlys) level of the diet, for each stage of production, is critical in setting the minimums for the other essential digestible amino acids, as they are defined as ratios relative to dlys. This formulation method is widely employed as it simplifies the leastcost process since a change in overall amino acid density only requires an alteration in the dlys level. An increase or decrease in amino acid density is often employed when significant changes are observed in either feed and/or meat prices; however, having a sound scientific basis for the degree of change in dlys, versus just an arbitrary adjustment, is critical in order to maximize profitability. As high priced ingredients, more limited resources and the environmental impact of excessive dietary nitrogen Several review papers, covering recent broiler amino acid research have been published (Tillman, 2011a, Tillman, 2012 and Tillman and Dozier, 2013). This paper, which is the 4th consecutive update on this topic, includes a thorough bibliography and set of tables, and picks up from the previous three papers by providing an updated review of digestible amino acid requirements and ratios to dlys through inclusion of the most recently published or presented information. Some additional emphasis will be placed upon digestible methionine (dmet) and digestible sulphur amino acids (dsaa: digestible methionine plus

2 digestible cysteine) as they were not covered in the first two review(s). Recent research on the other key essential digestible amino acids, which have the potential to be most deficient in typical broiler diets: Lysine, Threonine (dthr), Valine (dval), Isoleucine (dile), Tryptophan (dtrp) and Arginine (darg) will be updated. Then, in summary some economic concepts for optimizing the dlys level for maximum profitability will be discussed. Digestible Lysine Requirements Sriperm (2011a,b) evaluated the dlys level which would maximize bird performance, as well as the use of various models using the dlys level which would maximize economic return and profitability. The profitability portion of this will be discussed in more detail in the economic section towards the end of this paper. A central composite rotatable design was employed evaluating a titration of the dlys level during the grower (15-35d) as well as the finisher (early:35-42d or late:35-49d) phase. It was noted that during the grower phase, the dlys level which maximized bodyweight gain, feed conversion and either breast meat weight or yield were, 1.126, and 1.135%, respectively, averaging 1.196%. During the 15-42d growout, which encompassed both the grower and early finisher periods, the optimal dlys levels for the grower and early finisher respectively were : & 0.996% for bodyweight gain, & 0.997% for liveweight, & 0.994% for carcass weight, & 0.981% for breast meat weight and & 0.920% for breast meat yield. On average, these were 1.192% during the grower phase and 0.978% during the early finisher phase to day 42. For the longer growout (15-49d), encompassing both the grower and late finisher periods, the optimal dlys levels for the grower and late finisher respectively were: & 0.972% for bodyweight gain, & 0.992% for liveweight, & 0.988% for carcass weight, & 0.995% for breast meat weight and & 1.004% for breast meat yield. On average, these were 1.091% during the grower phase and 0.990% during the late finisher phase to day 49. It should be noted that the average dlys level for the grower and early finisher (35-42d) phases were 1.192% and 0.978%, respectively; however, the average dlys level for the grower and late finisher (35-49d) phases were 1.091%and 0.990%, respectively. The longer grow out decreased the optimal dlys level during the grower (15-35d) phase, but increased

3 the optimal dlys level during the finisher phase, despite the birds grown for an additional 7 days. In conclusion, it was noted that the dlys which maximized performance (growth and processing) depended to some degree upon the length of the growout period. Dozier and Payne (2012) determined the dlys requirement of broilers from 1 to 15 days of age in two experiments. The first trial, during October of 2009, used 1,600 Ross x Ross 708 females and the second trial, during September of 2010, used Hubbard x Cobb 500 female broilers. Corn, soybean meal, peanut meal (10.14%) and poultry by-product meal (5%) diets were fed. Digestible lysine was titrated in each trial from 0.95% to 1.43% in 0.08% increments, so as to create 7 treatment diets. In experiment 1, using quadratic brokenline analysis, the dlys requirement, from 1 to 7 days of age, was shown to be and for bodyweight gain and feed conversion, respectively. In experiment 2, also using quadratic broken-line analysis, the dlys requirement, from 1 to 7 days of age, was shown to be for bodyweight gain. Analysis of the dlys requirement from 1 to 14 days of age in trial 1 indicated a quadratic broken-line breakpoint at 1.27 for bodyweight gain; whereas in trial 2, it was shown to be 1.18 for bodyweight gain and for feed conversion. It was concluded that these requirement estimates were higher than previous recommendations and research attributable to some degree to the improved feed conversion of these birds. Nasr & Kheiri (2012) in a 2x3 factorial design with 2 means of formulation (using total versus digestible amino acids) and with 3 planes of Lys nutrition (-10% of NRC, NRC and +10% of NRC). Male broilers from 1-42d were used across 30 floor pens, providing 6 replicates per treatment. It was noted that diets based upon digestible amino acids had significantly greater breast percentage versus the alternative method of formulation. In addition, the higher plane of Lys nutrition also yielded both greater carcass (+4.4%) and breast (+1.81%) percentages than the diets with lower levels of Lys. The diet with the highest dlys level provided significantly higher carcass, breast and thigh weights versus the other 9 treatments. Oliveira et al. (2013) reported on the lysine requirement of 8 to 21 day of age broilers using an all-vegetable Corn-Soybean meal based diet. A 5x2 factorial design was used with five levels of dlys and 2 methods of formulating (only using Corn, soybean Meal and added methionine (CSM) versus balancing with numerous industrial amino acids (IAA)). Typical

4 responses to graded levels of dlys were noted for lysine intake, body weight gain, fat deposition, feed conversion and protein deposition. In the CSM and IAA diets, the dlys levels of 1.30 & 1.40% were deemed to provide optimum performance, respectively. The CSM diet estimate of 1.30% for the optimum dlys for bodyweight gain was determined using the quadratic maximum, whereas feed conversion optimum was determined to be 1.28% using the linear broken-line model. The IAA estimate for dlys was really not determinable using any break-point analysis and the reported 1.40% estimate was based upon the highest level actually fed as the response was essentially linear. Siqueira et al. (2013) examined the dlys requirement of Cobb 500 male broilers from 1-8d and 8-22d using either the diet dilution or the diet supplementation method. A 5x2 factorial design was used with five points of dlys and the two formulation methods noted above. Six replicates were used for each point with each replicate pen having 20 birds each. In the starter (1-8d) phase, dlys levels from to were used, incremented by In the grower (8-22d) phase, dlys levels from to were used, incremented by Using the feed conversion response from the diet dilution technique, which was viewed as superior to the diet supplementation approach, the estimated dlys requirements determined as the quadratic maximum were reported to be and 1.187, for the two phases respectively. Digestible Methionine Requirements and dmet/dlys Ideal Ratio Recommendations and Digestible Methionine+Cysteine Requirements and dsaa/dlys Ideal Ratio Recommendations Methionine is the first limiting amino acid for broilers fed corn-soybean meal diets and sulfur amino acids are used for lean tissue accretion, feather formation, and methyl donation (Garcia and Batal. 2005). Sulfur amino acid (SAA) requirements of broilers have been estimated from 0 to 3 weeks of age (National Research Council. 1994; Lumpkins et al., 2007), but data are limited with digestible SAA (dsaa) to Lys ratios for the starting chick. The dsaa to Lys ratio has been reported as 72 from 8 to 22 d of age (Baker and Han, 1994)

5 while Knowles and Southern (1998) reported the dsaa to Lys ratio at 66 and 63 for BW gain and feed efficiency. Kalinowski et. al. (2003) reported that the total methionine requirement in 0 to 3 week old broiler chicks was 0.50%, regardless as to whether they were slow or fast feathering strains. However, it was noted that the Cysteine requirement was 0.39% for slow-feathering males versus 0.44% for fast-feathering males. These values would predict a SAA requirement of between 0.89% and 0.94% for the 0 to 3 week of age broiler, depending upon feathering rate. Rubin et al. (2007) examined the effects of both methionine and arginine dietary levels on the immunity of broiler chickens submitted to immunological stimuli. They noted that Met has four primary functions: 1) protein synthesis, 2) glutathione precursor, 3) synthesis of polyamines and 4) methyl donation. Bunchasak (2009) wrote a review on the role of Met in poultry production. It was noted that Met supplementation improves the immune response through both direct and indirect effects. Direct effects were noted as protein synthesis while indirect effects were noted through compounds produced from methionine, such as homocysteine, glutathione and taurine. As such, methionine deficiency can lead to both humoral and nonspecific cellular immunocompetencies. It was noted relative to the requirement that it is higher than the NRC (1994), decreases with age, but increases with age when expressed as a ratio to lysine. Geraert and Mercier (2010) discussed the role of amino acids beyond those of protein synthesis. In their discussion, two of the areas which were highlighted were immunity and antioxidant activity, particularly the role of Met and SAA in these areas. Methionine had been shown to improve the immune status of birds via increased antibody production. It was also noted that the conversion of methionine into either glutathione or taurine were both cited as having positive impacts upon reducing oxidative stress. Goulart et al. (2011) reported on the dsaa ratio of Cobb male broilers from 1 to 42 days of age. Four phases were evaluated: pre-starter (1-7d), starter (8-21d), grower (22-35d) and finisher (36-42d). It was determined that the dsaa requirement was 0.873%, 0.755%, 0.748% and 0.661%, corresponding to dsaa/dlys ratios of 71, 70, 76 and 72%.

6 In a recent study, Dozier et al. (2013a) and Dozier and Mercier (2013) examined the dsaa ratio to Lys in Hubbard Cobb male broilers from 1 to 14 days of age. Eight concentrations of dsaa/dlys were fed ranging from 0.56 to 0.86% in increments of 0.06%. Diets were formulated to contain 1.16% dlys. Digestible SAA/dLys ratio was determined to be 78 and 77 from 1 to 7 and 1 to 14 days of age, which is higher than requirements estimated from previous research. Rostagno et al. (2011) has reported a dsaa to Lys ratio of 72 for 1 to 21 day old broilers. Research conducted with the modern broiler having market weights from 2.0 to 3.0 kg is sparse (Baker at al., 1996). Furthermore, these studies determined an absolute requirement and not the total SAA ratio to Lys. The dsaa to Lys ratio has been reported as 75 from 20 to 40 days of age (Mack et al., 1999). Moreover, Rostagno et al. (2011) estimated the dsaa to Lys ratio as 73 for 21 to 56 day old broilers. Mehri et. al. (2012) examined the ideal ratios for both methionine and threonine to lysine, of Ross x Ross 308 male broiler chicks, from day 3 to day 16. Response surface methodology was employed using a central composite rotatable design. Bodyweight gain was maximized when dlys was 1.12% and dmet was 0.54%, corresponding to a dmet/dlys ratio of 48. Similar requirement optimums were noted for feed conversion with dlys at 1.13% and dmet at 0.53%, corresponding to ideal ratios for dmet/dlys of 47. Dozier et al. (2013b) examined the dsaa to Lys ratio of Ross Ross 708 male broilers from 42 to 56 days of age. Broilers were fed 9 experimental diets consisting of 8 concentrations of dsaa/dlys ratios ranging from 0.60 to 0.88 in increments of A standardized Lys digestibility assay was conducted with broiler chicks to determine amino acid digestibility of the basal diet. Standardized dsaa of the basal diet was determined to be 0.53%. Progressive additions of dsaa/dlys resulted in a significant linear effects for breast meat weight (P = 0.057) and yield (P = 0.035), but no treatment differences were observed for growth performance. Digestible SAA/dLys ratios were estimated at 76 and 74 for total breast meat weight and yield. These data indicated that dsaa/dlys ratios for modern broilers are higher for total breast meat yield than growth performance.

7 Digestible Threonine Requirements and dthr/dlys Ideal Ratio Recommendations Brito et al. (2013) reported on the dthr requirement in 1-7d and 8-21d of age male and female (straight-run) Ross 508 broilers. For bodyweight gain during the 1-7d phase, using either the linear broken line (LBL) or the Quadratic maximum (Qmax) model, the dthr requirement estimate was 0.77 and 0.79%, respectively. During the 8-21d phase, the estimated dthr requirements were 0.67 and 0.71% from the LBL and Qmax models, respectively. Other variables measured did not exhibit a significant response to increasing levels of dthr. While the LB model gave lower error terms, it is generally considered that this model under-estimates the requirement (Morris, 1983). From the published paper by Mehri et. al. (2012) noted above, both the ideal ratios for methionine and threonine to lysine were examined in 3 to 16 day old broilers. Bodyweight gain was maximized when dlys was 1.12% and dthr was 0.78%, corresponding to a dthr/dlys ratio of 70. Similar requirement optimums were noted for feed conversion with dlys at 1.13% and dthr at 0.75% of the diet, corresponding to an ideal ratio of dthr/dlys of 66. Star et. al. (2012) looking at the threonine requirement of Ross 308 male broiler chickens from either day 9 to 20 or day 9 to 27 in three experiments. A subclinical infection model was employed at days 9 and 14 by inoculation with Eimeria maxima and Clostridium perfringens. While lesion incidence, lesion severity or mortality were not impacted by the dthr/dlys ratio, there were responses noted in body weight gain and feed intake with increased dthr/dlys ratios of 69 and 67, respectively. It was also noted in trial 3 that infected birds had improved bodyweight gain and feed intake when provided a dthr/dlys ratio of 67 versus one of only 63, which was also noted to carry-over out to day 37. It was concluded that while an increased dthr/dlys ratio did not improve intestinal damage from a subclinical E. maxima and C. perfringens inoculation, there were noted improvements in bodyweight gain and feed intake. In addition, linear broken-line analysis of gain per feed from experiments 1 & 2 generated a break-point at a 68 dthr/dlys ratio.

8 Duarte et al. (2012) examined the dthr requirement of 22 to 42 day of age male Cobb broilers. Six dthr levels were used and a level of dthr of % was determined to be the requirement using a linear broken line model. It should be noted that while dthr/dlys ratio of was reported, this trial design was not setup to determine a ratio. The dlys level used, of % was essentially at or above the requirement for these 22-42d old broilers and it is imperative in ratio work that the dlys level be set below the requirement. Mejia et al. (2012a) evaluated the dthr/dlys ratio of Ross 708 male broilers from days of age, using simultaneous dlys and dthr titrations. A range from 0.70% to 1.15% for dlys and of 0.40% to 0.85% for dthr were used in the determination of the optimal ratio. For BW gain, optimal dlys was 1.09% and optimal dthr was 0.72%, yielding a dthr/dlys ratio of 66. Absolute requirements for dthr were shown to be 0.67%, 0.72% and 0.74% for feed consumption, FC and breast meat weight, respectively. The dthr for maximum breast weight, put against the dlys for maximum bodyweight gain would yield a 68 ratio for dthr/dlys. Meloche et al, (2013) presented on the dthr/dlys ratio in 1-14 days of age Hubbard x Cobb 500 male broilers. Eight titration diets were used with dthr as a % of the diet ranging from 0.62 to 0.86%. The dlys level of the treatment diets was set below the requirement at 1.13% and each treatment was replicated across 8 pens. The titration of the dthr/dlys ratio ranged from 55 to 76. Linear broken line analysis was used as an estimate of the optimal ratio, although it is known that this is not ideal for representing a population and tends to underestimate the requirement. The optimal dthr/dlys ratio was determined to be 70 and 68 for bodyweight gain and feed conversion, respectively. It was concluded that a minimum ratio of 68 was required. Wecke & Liebert (2013) published in the open access, non-peer reviewed journal on a new, although yet un-validated approach to determining individual amino acid ratios. The need for further validation of this approach was noted by Pastor et al. (2013). Using nitrogen balance experiments, along with nitrogen deposition and retention determinants, the slope (also noted as the efficiency parameter) of a linear line from a control diet (adequate in all amino acid levels) was used against the slope of a linear line from a diluted diet (deficient in the test amino acid) as a means of calculating a ratio. Three trials, using thirty-five birds in

9 each were raised in metabolism cages with either 5 or 7 birds per experimental diet. Two ages were examined (11-21d) and (25-35d) for total amino acid ratios for Thr, Trp, Arg, Ile and Val. Several data points were omitted from the final conclusion based upon the slope of the nitrogen retention curve being non-significant and an average across the three trials were reported, sometimes being represented by only one trial. Reported total amino acid ratios for Trp (19 & 17) and Arg (105) were somewhat similar to those reported in the literature for digestible ratios while the reported ratios for Thr, Ile and Val, tended to be significantly lower than those reported digestible ratios. It should be noted however that a comparison between total and digestible ratio can be difficult to make. Jiang et al. (2014) reported on the dthr/dlys ratio for Hubbard M99 x Cobb 500 male broilers from 21 to 35 days of age. Eight titration diets were used with dthr as a % of the diet ranging from 0.49 to 0.77%. The dlys level of the treatment diets was set below the requirement at 0.95% and each treatment was replicated across 8 pens. The titration of the dthr/dlys ratio ranged from 51.2 to Quadratic broken line analysis was used to estimate the optimal dthr/dlys ratio and it was determined to be 68 and 67 for bodyweight gain and feed conversion, respectively. It was concluded that this study pointed to the dthr/dlys ratio being higher than previously published for the particular age range evaluated. Digestible Valine Requirements and dval/dlys Ideal Ratio Recommendations Corzo et al. (2010) examined requirement estimates for dval and dile in corn, SBM, meat & bone meal based diets fed to Ross TP16 male broilers from day 28 to day 42. A PC diet was formulated to be adequate in all nutrients, while a negative control diet was formulated to be deficient in both dval and dile. The control diets were formulated at 0.99% dlys with the PC having 0.75% dval and 0.66% dile and the negative control (NC) diets being 0.10% points lower for both of these AA. This in effect dropped the ratio of dval/dlys from 75 to 65 and that of dile/dlys from 66 to 56 across the two control diets. Six additional treatment diets were created by adding L-Val and/or L-Ile back to the NC diet such that either 50% or 100% of the differences for dval and/or dile were recovered. There was a significant decline in performance in both BW gain and feed intake when the dval and dile levels were

10 decreased from the PC to NC diets. Bodyweight was recoverable when either L-Val alone or in combination with L-Ile were added back to the negative control diet. Feed conversion required both added L-Val and added L-Ile to show improvement. As had been reported before, breast meat yield responded to supplemental L-Ile (Kidd et al., 2000). It was deemed that in these diets, containing meat & bone meal, that live performance responded more to dval and was fourth limiting; whereas dile supplementation increased breast meat yield more. As such, a scenario of co-limitation likely existed between these two essential AA in regards to optimizing growth and meat yield. Corzo et al. (2011) reported on a practical trial designed to evaluate the inclusion of L- Val in diets fed to Ross TP16 broilers from 28 to 42 days of age. Two control diets were formulated to meet all nutrient requirements. A positive control (PC) diet contained added DL-Methionine, L-Lys HCl and L-Thr and served as the dilution diet in the dval titration, while an industry control (IC) diet contained these AA plus L-Val at 0.034% and represented a potential diet of the future. A summit diet was formulated using 0.13% added L-Val and was blended in various proportions with the PC diet to make four intermediate titration diets, with added L-Val in increments of 0.026%. The summit diet was formulated to maintain a 78 ratio between dval and dlys, while allowing the ratios on dile, darg and dtrp to drop as SBM was replaced with added L-Val. No differences in performance or processing variables measured existed between the PC & IC diets, indicating L-Val could be least cost formulated into a diet at 0.034%, successfully. Results from the titration of L-Val inclusion suggested that up to 0.052% L-Val is feasible in practical diets without significantly sacrificing bird performance or processing measurements. Dozier et al (2012) examined the interactive effects of dval and dile to dlys ratios to male Ross x Ross 708 male broilers from either 28 to 42d of age (trial 1) or from 26 to 40d of age (trial 2). Trial 1 used 10 experimental diets consisting of a positive control diet (1.0% dlys) and a 3 x 3 factorial arrangement of dval/dlys ratio (74, 78 or 82) and dile/dlys ratio (63, 68 and 73) ) with a reduced dlys level (0.95%). Trial 2 was similar to Trial 1 in that it also used a 3 x 3 factorial arrangement of dval/dlys ratio (74, 78 and 82) and dile/dlys (62, 67 and 72) at a reduced dlys level (0.92%); however, two control diets were fed one at the dlys requirement (1.02%) and the other at a reduced dlys level (0.92%). These were

11 included to assure that dlys was slightly deficient in the treatments, by comparing the two controls, as well as to compare back to the treatment diet with similar dlys, dval and dile levels. In experiment one, a slightly significant (p=0.045) dval/dile ratio interaction was noted for feed conversion and mortality. No significant main effects for broiler performance or carcass characteristics were noted however, indicating that perhaps the 74 dval/dlys ratio was adequate. In experiment 2, no interactions were noted and the main effects of dval ratio to dlys were significant for bodyweight, bodyweight gain, feed conversion and abdominal fat which were optimized at a dval/dlys ratio of 82. Interestingly enough however, was the response in breast meat yield which was also significant being maximized at the lowest dval/dlys ratio. Tavernari et al, (2013) determined the optimal dval/dlys ratio in Male Cobb 500 (fast feathering) broilers between 8 & 21d (Starter) and between 30 and 43d (Finisher) of age. Appropriately, the dlys level of the all-vegetable based treatment diets was reduced below the requirement, in this case by 7%. Six titration points of dval/dlys were used in each of the phases. In the Starter these ranged from 69 to 84 while in the Finisher phase the ranged from 70 to 85, in both cases being incremented by 3 ratio points. For the Starter phase, the dval/dlys ratio for bodyweight gain, using 95% of Qmax and the LBL was shown to be 77 and 79, respectively. The feed conversion ratio was optimized at a dval/dlys ratio of 75 and 76 using 95% of Qmax and the LBL model, respectively. For the finisher phase, 95% of Qmax model gave a dval/dlys ratio of 75and 77, for bodyweight gain and feed conversion, respectively and 75 and 74, when using the LBL model. Berres et al. (2011), using male Cobb 500 broilers, studied the dval requirement, as a % of the diet from 21 to 42 days of age. An all-vegetable, corn-soybean based diet was used throughout and seven points on the titration curve were used, with dval ranging from 0.71 to 0.97%, based upon analysis. The range of dval levels was achieved through the use of added L-Val with all other ingredients being kept constant. Results were analysed using 95% of Qmax as well as the LBL model to determine the dval requirement and since dlys was set at 1.10%, this was not designed to be a ratio trial although ratios of 77 and 76 were reported for bodyweight gain and feed conversion. Results from using 95% of the Qmax indicated that the dval requirement, as a % of the diet, was 0.85, 0.84 and 0.85% for bodyweight gain, feed

12 conversion and abdominal fat pad yield, respectively. The LBL model predicted the dval requirement for bodyweight gain, feed conversion and abdominal fat pad yield to be 0.82, 0.81 and 0.73, respectively. Other measured variables did not show a significant response to dval titration, as often observed for the first three limiting amino acids in broiler feeds (SAA, LYS and THR). Campos et al (2012) examined the ideal amino acid ratios for arginine, isoleucine, valine and tryptophan in male broilers from 7-21 and from 28-40d. The dlys used for each phase (1.08 & 0.98%) were set below the requirement while all other nutrients (except for dval) were at or above the requirement, since this was a ratio trial. Three points on the curve were used for the dval / dlys ratio for the two phases : 70, 75 and 80 and 71.5, 77, 82.5, respectively. The dval / dlys ratios used during the 7-21d phase showed linear responses for bodyweight gain and feed conversion. The optimal dile / dlys ratio for the 28-40d phase was deemed to be 76. From a review of recent publications, it appears evident that dval is clearly fourth limiting in all vegetable based broiler feeds with corn and/or wheat as the primary grain source (Tillman, 2011, Tavernari et al, (2013)). Digestible Isoleucine Requirements and dile/dlys Ideal Ratio Recommendations Dozier et al. (2011) reported on the results from a trial designed identically to Corzo et al. (2010), except for the use of poultry by-product meal rather than meat and bone meal. The greatest breast meat responses in weight or yield tended to occur at the highest dile levels, but often in conjunction with additional L-Val. It was deemed that in these diets, dile was likely fourth limiting, although it was closely followed by dval, again implying a scenario of colimitation between these two essential AA. These two trials, point to the importance of setting proper ratios or levels for both dval and dile. Mejia et al. (2011) evaluated the dile/dlys ratio of Ross 708 male broilers from 28 to 42 days of age. A PC diet was formulated at 1.00% dlys and set to be adequate in all essential amino acids, including dile, with a ratio of 67. Two treatment diets were formulated at 0.95% dlys, which is slightly below the requirement, to have either a dile/dlys ratio of

13 57.8 or 74.4, representing the basal and summit titration treatment diets, respectively. These two dies were blended so as to create 5 intermediate diets across the 7 point titration curve. It was concluded a ratio of 68.9 was adequate for live performance and that a ratio of 71.7 gave similar results in regards to breast meat yield as to the PC diet, even though the latter was ~ 2.25 percentage points higher in CP. These results support the possibility of there being a higher dile/dlys ratio requirement to maximize breast meat responses. As described in the dval section above, Dozier et al. (2012) examined the interactive effects of dval and dile in two trials, using Ross x Ross 708 males. In experiment 1, the main effects indicated that increasing the ratio of dile to dlys reduced abdominal fat weight while increasing breast meat yield (at least up to 68). There was no significant impact on broiler growth parameters from increasing the dile/dlys ratio in either trial 1 or 2. As in trial 1, results from trial 2 indicated a significant response to increasing the dile/dlys ratio in terms of improving breast meat yield (at least up to 67). Tavernari et al (2012) evaluated the dile / dlys ratio in male Cobb 500 broilers from 7-21 and from days of age. A titration range from 58 to 75.3, incremented by 3.5 points providing 6 treatment levels. Diets were based upon Corn, soybean meal, corn gluten meal and spray-dried plasma. Results were reported as linear broken line (break-point), Qmax (within the Figures) and also as 95% of Qmax and Quadratic broken-line (QBL) (both within the text). As such, a close reading of the paper is required to fully extract the results. As the LBL typically underestimates the optimal ratio, I will discuss here the Qmax, 95% of Qmax and QBL although the LBL results are also shown in Table 6. During the 7-21d period, the following Qmax, 95% of Qmax and QBL values respectively were reported as : 68, 65, 64 for bodyweight gain, 69, 66, 65 for feed conversion, breast weight and breast fillet weight and 70, 67, 66 for breast yield and breast fillet yield. During the 30-43d period, the following Qmax, 95% of Qmax and QBL values respectively were reported as : 70, 66, 64 for feed intake, 72, 68, 68 for bodyweight gain and 75, 72, 72 for feed conversion. It was concluded overall that the dile / dlys ratio recommendation was 66 from 7-21d and 68 from 3-43d.

14 Campos et al (2012) examined the ideal amino acid ratios for arginine, isoleucine, valine and tryptophan in male broilers from 7-21 and from 28-40d. The dlys used for each phase (1.08 & 0.98%) were set below the requirement while all other nutrients (except for dile) were at or above the requirement, since this was a ratio trial. Three points on the curve were used for the dile / dlys ratio, for the two phases : 60, 65, 70 and 58, 67, 76, respectively. The dile / dlys ratios used during the 7-21d phase showed linear responses for bodyweight gain and feed conversion. The optimal dile / dlys ratio for the 28-40d phase was deemed to be 69. Digestible Tryptophan Requirements and dtrp/dlys Ideal Ratio Recommendations Hsia et al. (2005) evaluated the effect of tryptophan on growth and carcass characteristics in Hubbard male broilers in a series of three trials and across various ages. Trial 1 was conducted from 35-56d of age, trial 2 from 21-49d of age and trial 3 from 14-42d of age. Levels of total tryptophan fed in the three trials were: trial %, 0.228% and 0.258%, trial % or 0.198% and trial %, 0.167% and 0.198%. The total lysine level content was 1.1%, 1.0% and 1.0% for trials 1, 2 and 3, respectively. In trial 1, no differences were noted between the three treatments for feed intake, bodyweight gain, feed conversion or carcass characteristics. It was noted in trial 2 feed intake was not different between the two treatment levels; however bodyweight gain, feed conversion and a few of the carcass characteristics were significantly different in the high versus low total tryptophan level fed. In trial 3, the lowest level of tryptophan fed gave the lowest bodyweight gain and poorest feed conversion. In addition, the lowest total tryptophan level fed also gave the poorest weights for breast, thigh and heart. Corzo (2012) evaluated the arginine and tryptophan ideal ratios in Ross x Ross 708 male and female broilers from 1 to 18 days of age. No differences were noted between the two sexes. For dtrp, five points across the titration curve were used ranging from 10 to 22 in increments of 3 ratio points. A value of 95% of the quadratic maximum response was used as the estimate for the ideal ratio. The determined ideal ratios for bodyweight gain, feed intake

15 and feed conversion were 18, 19 and 17, respectively. On average, the ideal ratio for dtrp/dlys was 18. Campos et al (2012) examined the ideal amino acid ratios for arginine, isoleucine, valine and tryptophan in male broilers from 7-21 and from 28-40d. The dlys used for each phase (1.08 & 0.98%) were set below the requirement while all other nutrients (except for dtrp) were at or above the requirement, since this was a ratio trial. Three points on the curve were used for the dtrp / dlys ratio, for the two phases : 15, 16, 17 and 14, 17, 20, respectively. No responses were observed from the dtrp treatments during the 7-21d phase. The optimal dile / dlys ratio for the 28-40d phase was deemed to be 76. The order of limitation for tryptophan and arginine should generally fall after those for dval and dile. The ratio for tryptophan tends to be in the range of 16 to 18 and rarely is a constraint, even though it should be monitored, particularly in diets containing high levels of corn plus corn distillers dried grains with solubles. Digestible Arginine Requirements and darg/dlys Ideal Ratio Recommendations As noted above, Corzo (2012) evaluated the arginine and tryptophan ideal ratios in Ross x Ross 708 straight-run broilers from 1 to 18 days of age. No differences were noted between the two sexes. For darg, five points across the titration curve were used ranging from 75 to 115 in increments of 10 ratio points. As before, a value of 95% of the quadratic maximum response was used as the estimate for the ideal ratio. The determined ideal ratios for bodyweight gain, feed intake, feed conversion and livability were 108, 106, 114 and 103, respectively. On average, the ideal ratio for darg/dlys was determined to be108. Mejia et. al. (2012) evaluated the darg/dlys ratio of male Ross 708 broilers from 28 to 42 days of age during a constant, elevated temperature. Corn, soybean meal, corn distiller dried grains with solubles, meat & bone meal based diets were used. Digestible arginine was titrated, using L-Arg, across 7 treatment diets from a ratio of 100 to 130, relative to dlys which was set below the requirement at 0.95%. Based upon performance and processing responses, it was determined that the darg/dlys ratio was no higher than 105. It was concluded from this paper that darg typically should not become a formulation constraint

16 unless sorghum/milo is the primary grain source in the diet. Nonetheless, it is proper and advisable to set a minimum darg/dlys ratio of at least 105 and perhaps upwards of 108 in later phase diets, although it naturally increases across phases. Campos et al (2012) examined the ideal amino acid ratios for arginine, isoleucine, valine and tryptophan in male broilers from 7-21 and from 28-40d. The dlys used for each phase (1.08 & 0.98%) were set below the requirement while all other nutrients (except for darg) were at or above the requirement, since this was a ratio trial. Three points on the curve were used for the darg / dlys ratio, for the two phases : 100, 105, 110 and 95, 105, 115, respectively. No responses were observed from the dtrp treatments during the 7-21d phase. The darg / dlys ratios used during the 28-40d phase showed linear responses for bodyweight gain and feed conversion. Neto et al. (2013) examined the darg/dlys ratio in Cobb 500 male broilers from 21 to 42 days of age under high environmental temperature. While they only examined a 105 and 140 ratio of darg/dlys, they noted that broilers provided the 105 ratio had better live performance, carcass weights, carcass yields, longer villi length and shallower crypt depths. In addition, when the birds were innoculated with an antigen, the lower darg/dlys ratio gave a better immune response. Economics Several papers have addressed the impact of amino acid levels on optimizing profitability (DeBeer (2009, 2010), Eits et al. (2005 a,b), Lemme (2005), Pack et al. (2003), Ziggers (2011)). Due to the extensive use of ideal amino acid ratios in formulation, most of the emphasis has been placed upon the economically optimal dlys level; however, Kidd et al. (1998) noted the optimal level of dthr which maximized profitability was near the dthr level which also maximized broiler performance (feed conversion) and processing (carcass composition) parameters. It is likely that this is the case for all of the essential amino acids that feeding near their requirement for performance is also close to the point which maximizes profitability. Afterall, improvements in bodyweight gain, feed conversion and carcass

17 characteristics (particularly breast meat weight and yield) are often noted for several of these amino acids. Sriperm (2011a,b) evaluated various models to determine the economically optimal dlys level which would maximize profitability from selling whole carcass or parts. These scenarios were evaluated across a wide range of both feed ingredient (dietary) costs as well as meat (carcass, breast, wings and leg quarters) prices. For example, one example evaluated Corn at ~$280/ton and Soybean Meal at ~ $385/ton prices, along with the carcass price at ~ $0.64/pound price, the optimal dlys level to maximize profitability was 1.09% during the grower (15-35d) and 0.90% during the late finisher (35-49d), using a Cobb-Douglas production function. These values are not dissimilar to those determined from the static : production approach cited above. If carcass price were higher than that noted above, then the optimal dlys level for profitability would increase and it was noted that the meat or carcass price was more of a driver for the optimal profitability than is feed price. Tillman and Sriperm (2011b) presented a paper looking at the difference between dlys requirements determined from static : production (growth and feed efficiency) estimates versus those determined from profitability which also incorporates dynamic : market process (feed costs and meat prices). Various prediction models were employed, including the line intercept of models, the dlys requirement for static : production variables were determined using an average value from a quadratic broken-line (QBL) model, a quadratic and linear twoslope broken line model, the intercept of the linear broken-line model and quadratic polynomial model and the intercept of the QBL and quadratic polynomial model. It was shown that the requirement for Cobb 700 males from 28-42d were 0.95%, 0.99%, 1.00% and 0.97% for bodyweight gain, feed conversion, carcass weight and breast meat weight, respectively. These average of these static : production dlys requirements was 0.978%. When dynamic : market assessment of the dlys requirement which optimizes profitability was determined, across a wide range of feed and meat prices, the results were similar for the point which was the best case scenario (low feed but high meat prices). For example, the dlys level which maximized profitability for the carcass at the best case scenario was 0.986% and that for maximizing profitability from the breast meat was 0.980%. What was of particular note however was the worst case scenario for each example, which only reduced the

18 dlys level for maximum profitability by 0.889% and 0.947% for carcass and breast meat return, respectively. These differences from best to worst case only decreased the dlys level for maximum profitability by and percentage points, respectively. These value differences are less than what is sometimes done within the broiler industry, based upon what is felt to be the correct decision thus pointing out the importance of making well informed decisions when it comes to overall amino acid density of broiler diets. Abstracts of presentations are available from Perryman et al. (2013, Trial 1) and Tillman et al. (2013, Trial 2) with Ross x Ross 708 male broilers grown to 42 days of age and Hubbard M99 x Cobb 500 male broilers grown to 35 days of age, respectively. Five treatments were offered ranging from a basal to a summit diets, with intermediates labelled as industry low, industry high and requirement diets. For the Ross broilers, weighted dlys levels, based upon dietary dlys level and feed intake, from 0.86% to 1.14% in increments of 0.07 percentage points were fed across the three phases (starter: 1-14, grower: and finisher: 29-42). In both trials, linear responses in carcass weight, carcass yield, breast weight, breast yield, drumstick weight, wing weight and thigh weight were noted to increasing dlys levels and intakes. Quadratic responses were also noted for carcass weight, breast weight and breast yield in trial 1, but only for drumstick, wing and thigh weights in trial 2. A weighted average dlys level of 1.07%, was determined as the point where return over feed cost was maximized after 42 days of growth with the Ross 708. For the second paper presented, using Hubbard M99 x Cobb 500 male broilers, grown to 35 days of age, the weighted dlys level ranged from 0.88% to 1.16%, again in increments of 0.07 percentage points. A weighted average dlys level of 1.02%, was determined as the point where return over feed cost was maximized after 35 days of growth with the Hubbard M99 x Cobb 500 male broiler. Summary and Conclusions This paper has strived to provide an update on AA broiler nutrition during the past few years, with special emphasis on the dlys, dthr, dval, dile and darg requirements and more particularly the dthr/dlys, dval/dlys, dile/dlys and darg/dlys ratio recommendations.

19 With the enhancements in growth rates, FC and white meat yields, largely due to genetic selection, AA requirements have become critical to maximize technical performance while optimizing economic performance. The ratio on the next limiting AA, which cannot be supplemented, becomes highly critical in maintaining a proper nitrogen pool from which nonessential amino acids and proteins can be synthesized. Depending upon the ingredients used and the commercial AA s economically available, this next limiting AA can vary. Therefore, special emphasis needs to be placed on setting proper ideal ratios for these potentially limiting essential AA s (dval, dile, darg and dtrp) which are also required to optimize broiler performance and profitability.

20 NOTE: Abbreviations used within the following tables : 1BW=Bodyweight, BWG=Bodyweight gain, FC=Feed Conversion, FI=Feed Intake, CW=Carcass Weight, CY=Carcass Yield, BMW=Breast Meat Weight, BMY=Breast Meat Yield, BFW=Breast Fillet Weight (Pectoralis major),bfy=breast Fillet Yield, PA=Protein Accretion, VA=Valine Accretion, AFY =Abdominal Fat Yield. 2LBL=Linear Broken Line, QBL=Quadratic Broken Line, Q=Quadratic, Qmax = Quadratic Maximum, CCRD=Central Composite Rotatable Design / Response Surface. 3Veg.=Vegetable based diet, ABP=Animal by-product included in diet, BC = Blood Cells in the diet. Table 1. Digestible Lysine Requirements

21 Reference Strain Sex Age Parame dlys Method 2 Comment ter 1 (%) Dozier & Ross 708 F 1-7d BWG 1.35 QBL Payne (2012) 1-14d FCR BWG Hubbard x F 1-7d BWG 1.27 Cobb d BWG FCR Siqueira et Cobb 500 M 1-8d FCR Qmax Diet dilution al., (2013) 8-22d technique. Oliveira et Cobb M 8-21d BWG 1.30 Qmax al. (2013) FC 1.28 LBL Dozier et Ross TP16 M 14-28d BWG 1.09 QBL al. FC 1.15 (2009 a ) F BWG 0.98 FC 0.99 M BWG 1.07 x at 95% of FC 1.10 Qmax Dozier et al. (2009 b ) Sriperm (2011) Dozier et al. (2010 a ) F FC 1.03 Ross TP16 M 14-28d FC 1.18 x at 95% of Qmax BWG 1.23 LBL FI 1.22 FC 1.20 BWG 1.16 x at 95% of FC 1.20 Qmax BWG 1.18 LBL FC 1.24 Ross 708 M 15-35d BWG 1.13 QBL FCR 1.39 Ross TP16 M 28-42d BMW& 1.14 QBL BMY BWG 0.99 FC 1.05 CW 0.94 CY 0.92 BMW 0.96 Cobb 700 M 28-42d BWG 0.97 QBL FC 1.01 CW 1.03 CY 0.96 BMW 0.99 BMY 0.98 Humid Moderate

22 Shirley et al. (2009) Dimova et al. (2010) Cobb 500 Hubb M99xCob b500 M& F 35-49d BW 0.95 LBL BWG 0.96 FC 1.01 BFW 0.98 BMY 0.95 M 35-49d BWG 0.86 LBL FC 0.91 BMY 0.90 CW 1.02 Table 2. Digestible Methionine / dlys Ideal Ratio Recommendations Reference Strain Sex Age Parameter 1 dmet/dlys Method 2 Comment 3 Mehri et al. 3-16d BWG 48 CCRD (2012) FCR 47 Table 3. Digestible SAA / dlys Ideal Ratio Recommendations Reference Strain Sex Age Parameter 1 dsaa/dlys Method 2 Comment 3 Dozier et Hubbard M 1-7d 78 al. (2013 a ) xcobb d 77 Rostagno et 1-21d 72 al. (2011) 21-56d 73 Goulart et Cobb M 1-7d 71 QBL al. (2011) 8-21d d 76 Dozier et al. (2013 b ) 36-42d 72 Ross708 M 42-56d BMW 76 BMY 74 Table 4. Digestible Threonine / dlys Ideal Ratio Recommendations Reference Strain Sex Age Brito et al. (2013) Meloche et al. (2013) Mehri et al. (2012) Brito et al. (2013) Star et al. (2012) Ross 508 M&F 1-7d Hubbard x Cobb 500 M 1-14d Ross308 M 3-16d Ross 508 M&F 8-21d Ross308 M 9-20d Paramet dthr dthr/dly Method er 1 (%) s 2 Comment 3 BW 0.77 LBL Not a ratio BW 0.79 Qmax trial BWG 70 FCR 68 LBL BWG FCR CCRD BW 0.67 LBL Not a ratio BW 0.71 Qmax trial G/F LBL 69 QBL

23 Corzo et al. (2009) Jiang et al. (2014) Duarte et al. (2012) Wecke & Liebert (2013) Everett et al. (2010) Mejia et al. (2012) Kidd (1999) Dozier et al (2000) Ross TP16 Hubbard x Cobb 500 Cobb 500 Ross 308 Ross TP16 Ross708 Ross x Hubbard Ross308 M M M M M M M M 14-28d 21-35d 22-42d 25-35d 28-42d 35-49d 42-56d 42-56d BWG FCR BWG 68 FCR 67 FCR 0.76 Slope of N Retentio n Curves Reported a 71 ratio 62 General LBL QBL LBL AA Efficien cy BWG FCR QBL BMW Growth % of Carcass 0.67 Qmax BWG 0.68 FCR 0.67 CY 0.75 BMW 0.70 Not a ratio trial Total AA. Non-peer reviewed. Table 5. Digestible Valine / dlys Ideal Ratio Recommendations Reference Strain Sex Age Parameter 1 dval/dlys Method 2 Comment 3 Tavernari et Cobb 500 M 8-21d BWG 77 x at 95% Veg. al. (2013) FCR 75 of Qmax BWG 79 LBL FCR 76 Thornton et Ross 508 M 21-42d Multiple 68 ABP al. (2006) Corzo et al. Ross 708 M 21-42d BMY 72 x at 95% Veg. (2007) BWG 78 of Qmax BFW 77 BMW 77 BFW 74 BMY 74 Costa et al. (2010a) Wecke & Liebert (2013) Cobb d Multiple 76 x at Qmax Veg. Ross 308 M 25-35d Slope of N Retention Curves 79 AA Efficiency Total AA. Non-peer reviewed. Campos et al. Cobb 500 M 7-21d Multiple 78 x at Qmax Veg.

24 (2009 b ) 28-40d 79 Dozier et al. Ross 708 M 26-40d 82 (2012) 28-42d 74 Campos et al. (2012) Unknown M 28-40d Multiple 76 95% of Qmax Complex ABP Diets Tavernari et Cobb 500 M 30-43d BWG 75 x at 95% Veg. al. (2013) FCR 77 of Qmax BWG 75 LBL FCR 74 Costa et al. (2010 b ) Cobb d Multiple >77 x at Qmax Veg. Table 6. Digestible Isoleucine / dlys Ideal Ratio Recommendations Parameter dile/ Reference Strain Sex Age 1 Method 2 Comment 3 dlys Campos et al. Cobb 500 M 7-21d Multiple 67 x at Qmax (2009 a ) 28-40d 70 Tavernari et Cobb 500 M 7-21d BWG 68 Qmax. 95% Veg. al. (2012) & FC, BMW, 69 of value Helmbrecht et BFW reported in the al. (2010) BMY, BFY 70 text. BWG, FC, 62 LBL BMW, BMY, BFW BFY d BWG 63 FI 62 FC 69 BWG 72 Qmax. 95% FI 70 of value FC 75 reported in the text. Wecke & Liebert (2013) Campos et al. (2012) Mejia et al. (2011) Dozier et al.(2012) Ross 308 M 25-35d Slope of N Retention Curves 65 AA Efficiency Total AA. Non-peer reviewed. Unknown M 28-40d Multiple 69 95% of Qmax Complex ABP Diets Ross 708 M 28-42d FC 69 x at 95% of ABP BMY 72 Qmax Ross 708 M 26-40d BMY d 68

25 Table 7. Digestible Tryptophan / dlys Ideal Ratio Recommendations Reference Wecke & Liebert (2013) Campos et al. (2012) Corzo (2012) Strain Ross 308 Unkno wn Ross70 8 Se Age x M 11-21d 25-35d M 28-40d M 1-18d Parame dtrp/d ter 1 Lys Method 2 Comment 3 Slope of 19 AA Total AA. Non-peer N Efficiency reviewed. Retentio n Curves 17 Multiple 18 95% of Complex ABP Diets Qmax BWG 18 95% of FI 19 Qmax FC 17 Table 8. Digestible Arginine / dlys Ideal Ratio Recommendations Reference Strain Sex Age Corzo (2012) Wecke & Liebert (2013) Neto et al. (2013) Mejia et al.(2012) Ross7 08 Ross 308 Cobb5 00 Ross7 08 M &F M 1-18d 11-21d & 25-35d Parame darg/d ter 1 Lys Method 2 Comment 3 BWG 108 FI 106 FC % of Qmax Livabilit y 103 Slope of N Total AA. Nonpeer reviewed. Retentio 105 AA Efficiency n Curves M 21-42d 105 M 28-42d FC 105 LSD with =0.05 ABP

26 Table 9. Recommendations for Broiler Ideal Digestible Amino Acid Ratios relative to Digestible Lysine Minimum Ratios to Digestible Lysine = dmet/dlys dsaa/dlys dthr/dlys dval/dlys dile/dlys dtrp/dlys darg/dlys ME (Kcal / Lb) 1, Phase Start day End day Digestible Lysine (dlys) dmet dsaa dthr dval dile dtrp darg Pre-Starter % 0.59% 0.98% 0.91% 0.98% 0.86% 0.21% 1.37% Minimum Ratios to Digestible Lysine = dmet/dlys dsaa/dlys dthr/dlys dval/dlys dile/dlys dtrp/dlys darg/dlys ME (Kcal / Kg) 1, Phase Start day End day Digestible Lysine (dlys) dmet dsaa dthr dval dile dtrp darg Starter % 0.54% 0.90% 0.84% 0.90% 0.79% 0.19% 1.26% Minimum Ratios to Digestible Lysine = dmet/dlys dsaa/dlys dthr/dlys dval/dlys dile/dlys dtrp/dlys darg/dlys ME (Kcal / Kg) 1, Phase Start day End day Digestible Lysine (dlys) dmet dsaa dthr dval dile dtrp darg Grower % 0.51% 0.84% 0.76% 0.84% 0.74% 0.18% 1.17% Minimum Ratios to Digestible Lysine = dmet/dlys dsaa/dlys dthr/dlys dval/dlys dile/dlys dtrp/dlys darg/dlys ME (Kcal / Kg) 1, Phase Start day End day Digestible Lysine (dlys) dmet dsaa dthr dval dile dtrp darg Finisher % 0.47% 0.77% 0.68% 0.77% 0.68% 0.16% 1.07% Minimum Ratios to Digestible Lysine = dmet/dlys dsaa/dlys dthr/dlys dval/dlys dile/dlys dtrp/dlys darg/dlys ME (Kcal / Kg) 1, Phase Start day End day Digestible Lysine (dlys) dmet dsaa dthr dval dile dtrp darg Withdrawal % 0.43% 0.70% 0.61% 0.70% 0.62% 0.14% 0.97%