A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements in fulfilment of the degree for Master of Science. October 2018 / Glutathione S-transferases (GSTs) are Phase II detoxification enzymes that catalyse the conjugation of glutathione (GSH) to non-polar xenobiotic compounds to form water-soluble metabolites. Despite the low level of sequence similarity, the different GST classes follow the same canonical fold. hGSTP1-1 belongs to the Pi class and is involved in detoxification, as well as other non-classical roles such as regulating the MAP kinase pathway, protecting cells from nitrosative stress and regulating the function of 1-Cys peroxiredoxin. The structure, function and stability of GSTP1-1 was characterised to gain a better understanding of the general characteristics of the enzyme. The heterologous expression of hGSTP1-1 in Escherichia coli produces high yields of the enzyme that is then purified using immobilised metal affinity chromatography. A GSH-CDNB conjugation assay shows that the enzyme catalyses this reaction with a specific activity of 55.5 μmol/min/mg. The enzyme also binds 8-anilinonaphthalene-1-sulfonic acid (ANS), resulting in a blue shift and a two-fold increase in the fluorescence intensity of ANS. Far-UV circular dichroism shows that hGSTP1-1 is a predominantly alpha-helical protein, while intrinsic fluorescence studies show that the enzyme has Trp residues. Studies done using size exclusion HPLC show that the protein adopts a monomeric structure when exposed to high salt concentrations. Thermal unfolding of hGSTP1-1 shows that the enzyme unfolds irreversibly when exposed to increasing temperatures. Urea denaturation of the enzyme follows a two-state model (N2 ↔ 2U) and shows that domain 1 and domain 2 unfold in a cooperative manner. / E.R. 2019

Recent research suggests that amino acids can significantly increase skeletal muscle glucose uptake. However, the mechanism(s) have not been fully elucidated and it is also not clear if the beneficial impact amino acids have on healthy tissue translates to insulin resistant skeletal muscle. Therefore, in this series of studies, the effects of an amino acid mixture on glucose tolerance and insulin resistance were investigated. Study 1 Experiment-1 (Exp-1) demonstrated that an amino acid mixture significantly reduced the blood glucose response to an oral glucose challenge in Sprague Dawley rats. In Study 1 Exp-2, it was found that the improved glucose tolerance was due to an increase in skeletal muscle glucose uptake. The enhanced amino acid induced muscle glucose uptake was associated with improved cellular signaling. In Study 1 we could not determine the combined and/or individual effects of insulin and amino acids on glucose uptake, so in Study 2, the hindlimb of Sprague Dawley rats were perfused with glucose with or without amino acids in the presence and absence of insulin. Study 2, confirmed our previous findings that an amino acid mixture increased skeletal muscle glucose uptake compared to a carbohydrate supplement in the presence of insulin. The enhanced amino acid-stimulated glucose uptake was not due to increased phosphatidylinositol 3-kinase (PI 3-kinase) activity, although it was related to an increase in Akt substrate of 160 kDa (AS160) phosphorylation and a greater number of glucose transporters at the plasma membrane. In the final experiment, Study 3 investigated whether amino acids could improve glucose tolerance in an insulin resistant model. Study 3 Exp-1, demonstrated that an amino acid mixture significantly lowered the blood glucose response to an oral glucose challenge in obese Zucker rats. Study 3 Exp-2 showed that the improved glucose tolerance was due to enhanced amino acid induced skeletal muscle glucose uptake. Taken together, the results of this research suggests that adding an amino acid mixture to a carbohydrate supplement improves the blood glucose response to an oral glucose challenge, acutely lowers insulin resistance and this appears due to increased skeletal muscle glucose clearance and enhanced cellular signaling. / text

Previous work in many laboratories has established that the addition of L-valine to a culture of Escherichia coli K-12 growing exponentially on minimal medium causes inhibition resulting in persistent linear growth. The inhibition can be removed by addition of isoleucine. Growth of other strains of E. coli e.g. E. coli B and two mutants of K-12 (E. coli AB1020 and AB1005) was not affected by addition of valine. E. coli LL5, another mutant of K-12, showed no inhibition of growth by valine added in concentrations up to 1 x 10ˉ⁴M. However, the addition of 1 x 10ˉ³M valine caused a decrease in the logarithmic growth rate, and 1 x 10ˉ²M valine caused persistent linear growth. The inhibition of growth by valine in LL5 was antagonized by L-isoleucine (as is the case with wild-type E. coli K-12), but not by α-ketobutyrate, threonine or pyruvate. Kinetic studies showed that the maximal inhibition of acetohydroxy acid synthetase (AHAS) by 1.5 x 10ˉ³ M valine was 84% for E. coli K-12, 55% for E. coli B, 75% for E. coli AB1005, 77% for E. coli AB1020 and only 20% for E. coli LL5. It is proposed that the persistence of linear growth of K-12 in valine-containing medium is the result of incomplete (84%) feed-back inhibition of AHAS. Analysis of the data for valine inhibition of AHAS was carried out by the method of Levitsky and Koshland (1969) using Hill Plots. E. coli strains K-12, AB1005 and AB10 20 showed "positive cooperativity" between inhibitor (valine) binding sites at low valine concentrations, and "negative cooperativity" between inhibitor binding sites at high valine concentrations. The AHAS of E. coli strains E and LL5, however, showed only negative cooperativity between binding sites for inhibitor, which could be the mechanism for incomplete inhibition of AHAS by valine. Preliminary kinetic analyses using Michaelis-Menten plots were carried out with strains K-12 and LL5. The levels of threonine deaminase (TD) and AHAS were also examined in four of the E. coli strains, K-12, B, AB1020 and LL5. AHAS was repressed and TD derepressed in E. coli K-12 grown (linearly) with 10ˉ³M valine. In E. coli strains B and AB1020, growth with 10ˉ³M valine had no effect on the levels of AHAS or (derepressed) TD. In E. coli strain LL5, growth with 10ˉ³M valine did not change the AHAS level but caused significant derepression of TD. Sensitivity of growth to valine has been correlated with three properties of the organism: 1) Feed-back sensitivity of acetohydroxy acid synthetase, to L-valine; 2) The level of acetohydroxy acid synthetase in the cell; 3) The level of biosynthetic threonine deaminase - the regulatory enzyme for isoleucine biosynthesis. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Master of Science / Department of Animal Sciences and Industry / Joel DeRouchey / Michael Tokach / Six experiments using a total of 2,974 nursery pigs were used to determine the effects of monosodium glutamate (MSG) and amino acids (AA) on nursery pig growth performance. Experiments 1 and 2 evaluated increasing dietary MSG for nursery pigs. Increasing dietary MSG up to 2% without balancing for sodium and chloride content decreased nursery pig performance, and feeding sodium levels equivalent to 1% MSG also decreased performance. When sodium and chloride were balanced, there were marginal effects of increasing dietary MSG on pig performance. Experiment 3 was conducted to determine the standardized ileal digestible (SID) lysine (Lys) requirement for pigs weighing 7- to 11- kg. The SID Lys requirement was estimated to be 1.45% and greater than 1.60% depending on the statistical model applied for both ADG and G:F. This experiment served to validate the SID Lys requirement for use in formulating diets for the subsequent experiments. Experiment 4 evaluated increasing SID valine (Val) to Lys ratio for nursery pigs weighing 7- to 10- kg. A SID Val:Lys ratio of 62.9% optimized ADG. Maximum feed efficiency (G:F) was captured using 71.7% SID Val:Lys ratio, however, 99% of maximum was achieved with SID Val at 64.4% of Lys. For ADFI, maximum performance was at 74% SID Val:Lys ratio, with 99% of maximum intake achieved at 68%. Experiments 5 and 6 investigated increasing SID isoleucine (Ile) to Lys ratio for 6- to 11- kg pigs. When ADG and ADFI were modeled, broken-line models reported maxima of 52.0% Ile:Lys ratio while quadratic models were as high as 64% of Lys.

Student Number : 0002482E - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Proteins in the glutathione transferase family share a common fold. The close packing of secondary structures in the thioredoxin fold in domain 1 forms a compact hydrophobic core. This fold has a bababba topology and most proteins/domains with this fold have a topologically conserved isoleucine residue at the N-terminus of a-helix 3. Class Alpha glutathione transferases are one of 12 classes within the glutathione transferase family. To investigate the role of the conserved isoleucine residue in the structure, function and stability of glutathione transferases, homodimeric human glutathione transferase A1-1 (hGST A1-1) was used as a representative of the GST family. Ile71 was replaced with valine and the properties of I71V hGST A1-1 were compared with those of wildtype hGST A1-1. The spectral properties monitored using far-UV CD and tryptophan fluorescence indicated little change in secondary or tertiary structure confirming the absence of any gross structural changes in hGST A1-1 due to the incorporation of the mutation. Both wildtype and mutant dimeric proteins were determined to have a monomeric molecular mass of 26 kDa. The specific activity of I71V hGST A1-1 (130 mmol/min/mg) was three times that of wildtype hGST A1-1 (48 mmol/min/mg). I71V hGST A1-1 showed increased kinetic parameters compared to wildtype with a 10-fold increase in kcat/Km for CDNB. The increase in Km of I71V hGST A1-1 suggests the mutation had a negative effect on substrate binding. The DDG for transition state stabilisation was –5.82 kJ/mol which suggest the I71V mutation helps stabilise the transition state of the SNAR reaction involving the conjugation of reduced glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB). A 2-fold increase in the IC50 value for I71V hGST A1-1 (11.3 mM) compared to wildtype (5.4 mM) suggests that the most noticeable change due to the mutation occurs at the H-site of the active site. Conformational stability studies were performed to determine the contribution of Ile71 to protein stability. The non-superimposability of I71V hGST A1-1 unfolding curves and the decreased m-value suggest the formation of an intermediate state. The conformational stability of I71V hGST A1-1 (16.5 kcal/mol) was reduced when compared to that of the wildtype (23 kcal/mol). ITC was used to dissect the binding energetics of Shexylglutathione to wildtype and I71V hGSTA1-1. The ligand binds 5-fold more tightly to wildtype hGST A1-1 (0.07 mM) than I71V hGST A1-1 (0.37 mM). The I71V mutant displays a larger negative DCp than wildtype hGST A1-1 (DDCp = -0.41 kJ/mol/K). This indicates that a larger solvent-exposed hydrophobic surface area is buried for I71V hGST A1-1 than for wildtype hGST A1-1 upon the binding of S-hexylglutathione. Overall the results suggest that Ile71 conservation is for the stability of the protein as well as playing a pivotal indirect role in catalysis and substrate binding.

O objetivo deste estudo foi avaliar o desempenho produtivo, o rendimento de carcaça e de cortes comerciais de frango alimentados com rações peletizadas contendo diferentes relações de valina/isoleucina durante as fases de crescimento e final (22 a 42 dias) desses animais. Foram utilizados 630 pintos machos de um dia de idade, da linhagem cobb 500. De 1 a 22 dias de idade, todas as aves receberam uma ração basal comum e aos 22 dias foram pesadas e distribuídas em um delineamento inteiramente casualizado, em esquema fatorial 3x3 (nove combinações de níveis de valina e isoleucina). Os frangos de corte foram submetidos aos seguintes tratamentos experimentais: T1 ração com relação valina/lisina 0,75% e isoleucina/lisina de 0,67%; T2 ração com relação valina/lisina 0,75% e isoleucina/lisina 0,71%; T3 ração com relação valina/lisina de 0,75% e isoleucina/lisina de 0,75%; T4 ração com relação valina/lisina de 0,79% e isoleucina/lisina 0,67%; T5 ração com relação valina/isoleucina de 0,79% e isoleucina/lisina 0,71%; T6 - ração com relação valina/isoleucina de 0,79% e isoleucina/lisina 0,75%; T7 - ração com relação valina/isoleucina de 0,83% e isoleucina/lisina 0,67%; T8 - ração com relação valina/isoleucina de 0,83% e isoleucina/lisina 0,71% e T9 - ração com relação valina/isoleucina de 0,83% e isoleucina/lisina 0,75%. As características avaliadas foram desempenho, o ganho de peso, o consumo de ração, a conversão alimentar, o rendimento de carcaça e cortes comerciais. Houve interação entre os fatores para todas as características avaliadas, exceto para peso corporal aos 28 dias. Os melhores resultados obtidos nas características avaliadas foram para as aves alimentadas com (0,83% de valina e 0,75% de isoleucina), exceto para peso corporal e ganho de peso aos 42 dias, em que os melhores resultados foram obtidos pelas aves que receberam (0,83% de valina e 0,75% de isoleucina) / The objective of this study was to evaluate the performance, carcass yield and commercial cuts fed pelleted diets containing different ratios of valine / isoleucine during the growing and final (22-42 days) phase of these animals. I was used 630 one day old male cobb 500 broiler chicks during 1-22 days of age, the birds were fed a common basal diet at 22 days the birds were weighed and distributed in a completely randomized design with factorial 3x3 (nine combinations of levels of valine and isoleucine). Broilers were submitted to the following experimental treatments: T1 - ration with respect valine / isoleucine and lysine 0.75% / 0.67% lysine; T2 - ration with respect valine / isoleucine and lysine 0.75% / 0.71% lysine; T3 - ration with respect valine / lysine and isoleucine 0.75% / 0.75% lysine; T4 - ration with respect valine / lysine and isoleucine 0.79% / 0.67% lysine; T5 - tion with respect valine / isoleucine isoleucine and 0.79% / 0.71% lysine; T6 - tion with respect valine / isoleucine isoleucine and 0.79% / 0.75% lysine; T7 - tion with respect valine / isoleucine isoleucine and 0.83% / 0.67% lysine; T8 - tion with respect valine / isoleucine isoleucine and 0.83% / 0.71% lysine and T9 - tion with respect valine / isoleucine isoleucine and 0.83% / 0.75% lysine. The characteristics evaluated were weight gain, feed intake, feed conversion and carcass yield and animal commercial cuts. There was a significant interaction between the factors for all characteristics evaluated, except for weight at 28 days. The best results in characteristics were evaluated for birds receiving (0.83% of valine and isoleucine 0.75%), except for body weight and weight gain at 42 days, in which the best results were obtained the animals receiving (0.83% and 0.75% of valine for isoleucine)

O objetivo deste estudo foi avaliar o desempenho produtivo, o rendimento de carcaça e de cortes comerciais de frango alimentados com rações peletizadas contendo diferentes relações de valina/isoleucina durante as fases de crescimento e final (22 a 42 dias) desses animais. Foram utilizados 630 pintos machos de um dia de idade, da linhagem cobb 500. De 1 a 22 dias de idade, todas as aves receberam uma ração basal comum e aos 22 dias foram pesadas e distribuídas em um delineamento inteiramente casualizado, em esquema fatorial 3x3 (nove combinações de níveis de valina e isoleucina). Os frangos de corte foram submetidos aos seguintes tratamentos experimentais: T1 ração com relação valina/lisina 0,75% e isoleucina/lisina de 0,67%; T2 ração com relação valina/lisina 0,75% e isoleucina/lisina 0,71%; T3 ração com relação valina/lisina de 0,75% e isoleucina/lisina de 0,75%; T4 ração com relação valina/lisina de 0,79% e isoleucina/lisina 0,67%; T5 ração com relação valina/isoleucina de 0,79% e isoleucina/lisina 0,71%; T6 - ração com relação valina/isoleucina de 0,79% e isoleucina/lisina 0,75%; T7 - ração com relação valina/isoleucina de 0,83% e isoleucina/lisina 0,67%; T8 - ração com relação valina/isoleucina de 0,83% e isoleucina/lisina 0,71% e T9 - ração com relação valina/isoleucina de 0,83% e isoleucina/lisina 0,75%. As características avaliadas foram desempenho, o ganho de peso, o consumo de ração, a conversão alimentar, o rendimento de carcaça e cortes comerciais. Houve interação entre os fatores para todas as características avaliadas, exceto para peso corporal aos 28 dias. Os melhores resultados obtidos nas características avaliadas foram para as aves alimentadas com (0,83% de valina e 0,75% de isoleucina), exceto para peso corporal e ganho de peso aos 42 dias, em que os melhores resultados foram obtidos pelas aves que receberam (0,83% de valina e 0,75% de isoleucina) / The objective of this study was to evaluate the performance, carcass yield and commercial cuts fed pelleted diets containing different ratios of valine / isoleucine during the growing and final (22-42 days) phase of these animals. I was used 630 one day old male cobb 500 broiler chicks during 1-22 days of age, the birds were fed a common basal diet at 22 days the birds were weighed and distributed in a completely randomized design with factorial 3x3 (nine combinations of levels of valine and isoleucine). Broilers were submitted to the following experimental treatments: T1 - ration with respect valine / isoleucine and lysine 0.75% / 0.67% lysine; T2 - ration with respect valine / isoleucine and lysine 0.75% / 0.71% lysine; T3 - ration with respect valine / lysine and isoleucine 0.75% / 0.75% lysine; T4 - ration with respect valine / lysine and isoleucine 0.79% / 0.67% lysine; T5 - tion with respect valine / isoleucine isoleucine and 0.79% / 0.71% lysine; T6 - tion with respect valine / isoleucine isoleucine and 0.79% / 0.75% lysine; T7 - tion with respect valine / isoleucine isoleucine and 0.83% / 0.67% lysine; T8 - tion with respect valine / isoleucine isoleucine and 0.83% / 0.71% lysine and T9 - tion with respect valine / isoleucine isoleucine and 0.83% / 0.75% lysine. The characteristics evaluated were weight gain, feed intake, feed conversion and carcass yield and animal commercial cuts. There was a significant interaction between the factors for all characteristics evaluated, except for weight at 28 days. The best results in characteristics were evaluated for birds receiving (0.83% of valine and isoleucine 0.75%), except for body weight and weight gain at 42 days, in which the best results were obtained the animals receiving (0.83% and 0.75% of valine for isoleucine)

Chick feeding tests were conducted to study the effects of dietary imbalances among the three branched chain amino acids on growth rate. All diets fed in these tests contained about 18% protein. The indispensable amino acids (IAA) were found in two of the diets fed in the proportions found by Dobson et al. (1964) to be well balanced. One of the diets had all IAA at 85% of these balanced levels while the other had them all at 125% of these levels. The tests involved reducing the isoleucine, leucine and valine levels in the high IAA diet to the low levels in all combinations. Growth rates were similar with the diets containing all ten IAA at the low levels, all ten at the high levels, or seven at the high levels and isoleucine, leucine and valine at the low levels. Generally, when only one or two of these three were reduced to the low levels, growth rates were lower. Thus there appeared to be a three-way interaction among these amino acids with the reduction in weight gain being the result of an imbalance and not of a deficiency. The changes in growth rate noted when the leucine level was reduced indicated that the leucine level in Dobson's balanced diet was relatively high; the isoleucine level appeared to be relatively low. The interaction between leucine and valine appeared to be more significant than the other two two-way interactions. The effect of dietary level of these amino acids on the branched chain amino acid transaminase (BAT) activity in the liver and kidneys was determined. The differences noted were inconsistent. There tended to be a slightly higher activity in chicks fed the high levels of these amino acids, but the differences certainly were not as great as the changes in arginase activity reported by others when imbalanced diets were fed. Chicks fed the diet low in valine and high in the other nine IAA were selected for fast or slow growth on this diet. They were raised to maturity and produced eggs that were hatched for feeding tests with the diets containing different levels of the branched chain amino acids. Only a limited number of chicks from the two strains were hatched. Performance of the chicks was similar to that of their parents when fed the low valine diet.

Within commercial broiler production, feed and feed manufacturing costs account for 60 to 70% of production costs. Of these feed costs specifically, ingredients that supply energy and protein represent the highest expenditure, verifying the importance of knowing the nutrient requirements of current broiler strains to ensure accurate least-cost formulation. To reduce these production costs and optimize performance, primary breeder companies and nutritionists continuously strive to improve nutrient utilization of commercial broiler strains. As a result of this, a great magnitude of work is conducted to determine the nutrient requirements for broilers, especially amino acids. Therefore, the overall objective of this dissertation was to utilize processing measurements, multiple statistical models, and economics as a whole to determine the optimal percent digestible Isoleucine:digestible Lysine ratio (dIle:dLys) for Ross 708 x Ross YP male broilers to improve growth performance and economic return. Chapter 2 determined the optimal dIle:dLys ratio for the starter phase (d 0-18), while Chapter 3 determined the optimal ratio for the grower phase (d 14-28), and Chapter 4 determined the optimal ratio for the finisher phase (d 28-42). All experimental diets for each respective growth phase were created from a common deficient corn and soybean meal-based diet. After manufacturing, half was retained to create the summit diet through the addition of crystalline Ile. The remaining five experimental diets for each respective growth phase were obtained by blending proportions of the deficient and summit diets. All dIle:dLys ratios were estimated using quadratic regression (QR; 95% of the asymptote), as well as linear and quadratic broken line models (LBL; QBL). Regression analysis from d 0-18 using multiple regression models estimated the ratios to range from 63-73% for BW and BWG and 68-74% for FCR. Based on the data from the grower phase and using multiple regression models, the estimated ratios ranged from 62-68% for BW and BWG and 67-70% for FCR and are similar to the current breeder nutrient specifications. Data from the finisher phase and once again using multiple regression models estimated the ratios to range from 62-66% for BW and BWG and 63-66% for FCR.

Esta tese foi realizada objetivando comparar 4 programas alimentares, em dietas à base de milho e farelo de soja, formuladas com ou sem restrição de proteína bruta (PB), suplementadas ou não com L-Val e L-Ile, e usando diferentes níveis de lisina digestível (dig.). Os programas alimentares (PRG) foram: PRG 1, PB limitada a um mínimo (22,4, 21,1, 19,8, 18,4% para as fases pré-inicial, inicial, crescimento e final, respectivamente, com relações de AA:Lis definidos apenas para Met+Cis (0,72) e Tre (0,65); PRG 2, a PB não foi restrita e as relações estendidas para Val (0,77) e Ile (0,67); PRG 3, mesmas restrições do PRG 2 e suplementadas com L-Val; PRG 4, mesmas restrições do PRG 3, e suplementadas com L-Ile. Dois experimentos foram realizados, um com 1.800 e outro com 4.800 pintos machos Cobb x Cobb 500 de 1 dia de idade. No primeiro experimento, as rações foram formuladas usando os 4 PRG e níveis de Lis dig. de 1,324%, 1,217%, 1,095% e 1,006% ou 5% maiores, para cada fase, totalizando 8 tratamentos e 9 repetições cada. Para o segundo experimento, os 4 PRG foram utilizados até 1-21 d, totalizando 4 tratamentos com 48 repetições. E de 22 a 42 dias, cada PRG foi subdividido nos 4 PRG, passando cada PRG da fase anterior a receber todos os 4 PRG para as fases de crescimento e final, totalizando 16 tratamentos com 12 repetições cada. Não houve interação entre os tratamentos em ambos os experimentos, com exceção para o ganho de peso (GP) e conversão alimentar (CA) de 36 a 43d, no primeiro experimento, onde aves alimentadas com PRG 2 demonstraram melhorias no GP e CA quando alimentadas com um aumento de 5% no nível de Lis. Também, o GP e a CA acumulados aos 35 e 43d foram melhores quando as aves foram alimentadas com o PRG 2, sem diferença estatística para o PRG 3 e 4. Um aumento em 5% no nível de Lis dig. resultou na melhoria da CA acumulativa aos 43d. A gordura abdominal, como porcentagem da carcaça eviscerada aos 43d, foi maior para as aves alimentadas com a PRG 1. No segundo experimento, as aves alimentadas com PRG 2 tiveram melhor CA de 22 a 42d e de 1 a 42d, mas sem diferença estatística para os PRG 3 e 4. As aves alimentadas com PGR 1 de 1 a 42d apresentou o menor GP e o pior CA sem diferenças entre os PGR 2 e 3, e os PGR 3 e 4, respectivamente. Os dados do presente estudo demonstram que a suplementação de dietas para frangos de corte com fontes sintéticas dos cinco primeiros AA limitantes permitiram resultados de desempenho semelhantes a uma dieta com PB restrita a um valor mínimo. / This thesis was carried out to compare four corn-soy feeding programs formulated with or without crude protein (CP) restrictions supplemented with or without L-Val and L-Ile, and using different digestible (dig.) Lys levels. Feeding programs (PRG) were: PRG 1, CP was restricted to a minimum (22.4, 21.1, 19.8, 18.4% for pre starter, starter, grower and finisher phases, respectively) with AA to Lys ratios set only for TSAA (0.72) and Thr (0.65); PRG 2, CP was not restricted while minimum ratios were also extended to Val (0.77) and Ile (0.67); PRG 3, restrictions were as in PRG 2 but with L-Val added; PRG 4, restrictions were as in PRG 3, but with L-Ile added. For this two experiments were conducted, one using 1,800 and the other using 4,800 oneday- old Cobb 500 male broiler chicks. For the first experiment, feeds had formulated using the 4 PRG and dig. Lys as 1.324%, 1.217%, 1.095% and 1.006% or 5% higher, for each phase, totaling 8 treatments with 9 replicates each. For the second experiment, the 4 PRG were used from 1 to 21 d, totaling 4 treatments with 48 replicates. From 22 to 42d, each PRG was subdivided in 4 PRG having the same rational as it was done to 21d, where each of the 4 PRG began receiving all 4 PRG for grower and finisher phases, totaling 16 treatments with 12 replicates each. No interaction was found between treatments in both experiments, with one exception for body weight gain (BWG) and feed conversion ratio (FCR) from 36 to 43d, in the first experiment, with birds fed PRG 2 demonstrating improvements in BWG and FCR when fed the 5% increasing in dig. Lys. And also, cumulative BWG and FCR at 35 and 43d and in each individual feeding phases showed broilers from PRG 2 having the best BWG and FCR; however, mean separations using Tukey showed no difference from birds fed PRG 3 and 4. Feeding a dietary program with 5% increase in dig. Lys resulted in improvement in cumulative FCR at 43d. Abdominal fat, as a percentage of the eviscerated carcass at 43d, was highest for birds fed the PRG 1. For the second experiment, birds fed with PRG 2 led to the best FCR from 22 to 42d and from 1 to 42d, but without statistical difference from PRG 3 and 4. Birds fed PGR 1 from 1 to 42d showed the lowest BWG and the highest FCR without statistical differences from PGR 2 and 3 and PGR 3 and 4, respectively. Data from the current study demonstrate that the supplementation of broiler diets with crystalline sources of the first five limiting AA allowed performance results similar to those produced when CP is restricted to a minimum.