Efficacy of guanidinoacetic acid on growth and muscle energy metabolism in broiler chicks receiving arginine-deficient diets

Efficacy of guanidinoacetic acid on growth and muscle energy metabolism in broiler chicks... Abstract Guanidinoacetic acid (GAA) is formed from arginine (Arg) and is the immediate precursor to creatine (Cr) and phosphocreatine (PCr), which are important compounds involved in muscle energy homeostasis. This study sought to determine whether GAA could spare Arg in broiler chicks fed an Arg-deficient practical diet. A basal [0.84% standardized ileal digestible (SID) Arg] was supplemented with combinations of L-Arg (0 or 0.16%) and GAA (0, 0.06, or 0.12%) to form a factorial arrangement of diets; the unsupplemented basal served as the negative control (NC). Additionally, the basal was supplemented with 0.32% Arg to generate an Arg-adequate positive control (PC). Mash diets were fed to 8 replicate pens of 5 chicks per treatment from d 8 to 22 posthatch, with measurements including growth performance, blood GAA metabolites, muscle cellular energy markers, and clinical outcomes. Supplementation of 0.16% Arg increased (P < 0.05) BW gain from d 15 to 22 posthatch, while graded addition of GAA tended to improve BW gain (P < 0.094). Supplementation of either Arg or GAA increased (P < 0.05) feed efficiency from d 15 to 22 and d 8 to 22 posthatch. Birds fed the PC diet had greater (P < 0.05) responses for nearly all blood and tissue outcomes compared with NC-fed birds. Serum GAA was more responsive to supplementation of GAA in the presence versus absence of supplemental Arg (interaction, P < 0.001). Interactions (P < 0.05) were also observed for concentrations of muscle total Cr, creatinine, and most serum essential amino acids, notably Arg. Serum Cr, as well as muscle PCr, total Cr, and glycogen were increased (P < 0.05) independently by Arg and GAA supplementation, with highest levels achieved via combined addition of 0.12% GAA and 0.16% Arg. Minimal effects were detected on hematological and clinical chemistry outcomes. Overall, we conclude that GAA supplementation can spare Arg in broiler chicks fed Arg-deficient practical diets as evidenced by improvements in growth performance and muscle energy stores. INTRODUCTION Volatility in commodity prices and variability in available nutrient content of alternative feedstuffs necessitate careful control over feed formulations for livestock. With increased use of alternative protein sources and a move to lower crude protein concentrations to achieve financial and environmental benefits, the need to incorporate crystalline amino acids (AA) to maintain optimal dietary profiles is rising. Arginine (Arg) is generally considered either the fourth or fifth limiting AA for broiler chickens (Han et al., 1992; Fernandez et al., 1994; Waguespack et al., 2009), but is not currently available in an economically viable form for the animal feed industry. Lower crude protein formulations, increased use of co-product and by-product ingredients such as distillers’ dried grains with solubles (DDGS) (Parsons and Baker, 1983), and a reduction of animal derived protein sources in feed formulations all result in decreased inherent Arg concentrations in the diet of broilers. In contrast, increased growth rate of modern broilers (Havenstein et al., 2003), and a lack of de novo synthesis of Arg (Tamir and Ratner, 1963a), both mandate higher dietary Arg requirements. Therefore, additional dietary Arg sources may be substantial to optimize broiler production in the near future (Han et al., 1992). Arg is an essential AA for broiler growth, but is classified as well as functional AA due to its other roles for broiler nutrition and health (Wu and Morris, 1998). Considering the essentiality of dietary Arg to support lean tissue accretion in the broiler and the lack of a commercially available source of Arg, dietary strategies fueling other metabolic fates of Arg (i.e., for non-protein functions) might be promising solutions to spare Arg, thereby allowing a greater proportion of this AA to be used for muscle protein synthesis. All vertebrate animals generate guanidinoacetic acid (GAA) as an intermediate in the synthesis of creatine (Cr), with the metabolic pathway involving several tissues (Beitz, 2004). In the initial step, GAA is synthesized in the kidney from Arg and glycine (Gly) via Arg: Gly amidinotransferase (AGAT) (Wu and Morris, 1998), and GAA is subsequently imported from the circulation by the liver and converted to Cr. This step is catalyzed by guanidinoacetic acid methyltransferase (GAMT) using S-adenosyl-methionine as a methyl donor, hence producing S-andenosyl homocysteine as a side product (Brosnan et al., 2009; Ostojic et al., 2013). Synthesized Cr is subsequently exported from the liver and transported to target organs, primarily those tissues with high, fluctuating energy demand (i.e., skeletal muscle, heart, brain, retina, spermatozoa), where it is phosphorylated to phosphocreatine (PCr). PCr serves critical physiological functions both as a reserve of high-energy phosphate groups to restore adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and as part of the cellular energy transport system. Due to this central role in cell energy transfer, Cr is present in high concentrations in muscle tissue. However, tissues have a limited storage capacity for Cr and cannot be overloaded (Harris et al., 1992; Greenhaff et al., 1994), so high levels of circulating Cr induce a negative feedback loop on endogenous Cr synthesis by decreasing AGAT expression and thereby decreasing GAA synthesis, most likely in an effort to conserve Arg and methionine (Met) as essential AA for protein synthesis (Wyss and Kaddurah-Daouk, 2000). Creatine synthesis represents a sizeable proportion of whole-body Arg usage, hence providing dietary Cr through use of animal protein ingredients is one strategy to optimize tissue Cr stores, independent of endogenous Cr synthesis, and allow availability of Arg for other functions, namely protein synthesis. However, processed animal protein ingredients available in the feed industry contain low concentrations of Cr (Harris et al., 1997; Dobenecker and Braun, 2015), and crystalline Cr is neither commercially available nor stable through the standard feed manufacturing process (Baker, 2009).Therefore, GAA provided as a crystalline feed additive may be important not only for sparing Arg, but also as the immediate metabolic precursor to Cr, for maintaining overall energy homeostasis in the bird by supporting restoration of cellular ATP concentrations under conditions of high energy turnover (i.e., PCr maintains ATP homeostasis in myofibrils). The Arg-sparing capacity of GAA and Cr has been extensively studied (Edwards et al., 1958; Savage and O’Dell, 1960), but this work was conducted largely using either semi-purified or purified diets. More recent research (Dilger et al., 2013) indicates that GAA improves feed efficiency not only in semi-purified diets deficient in Arg, but also when supplemented in Arg-deficient practical diets. Because the only metabolic fate of GAA is the synthesis of Cr, the Arg-sparing effect of GAA may be due to the decreased need of Arg for GAA and subsequent Cr production. Additionally, there are no safety indications in published literature for dietary GAA fed to broilers. Based on the available evidence that GAA supplementation of Arg-deficient diets elicited an improved growth response, we sought to further investigate the dose-response relationship of GAA on growth performance, blood levels of relevant metabolites (e.g., Cr and Arg), safety outcomes based on clinical pathology and hematological measures, as well as muscle energy stores (ATP, PCr, Cr, and glycogen) when included in practical broiler chicken diets containing varying concentrations of Arg. MATERIALS AND METHODS All animal care procedures were approved by the University of Illinois Institutional Animal Care and Use Committee before initiation of the studies. Animals and Diets Two-hundred eighty male Ross 708 chicks (Hoovers Hatchery, Rudd, IA) were maintained in thermostatically controlled starter batteries with raised-wire floors in an environmentally controlled room with continuous lighting. Water and experimental diets were provided on an ad libitum basis throughout the study. Chicks arrived at 2 d posthatch and received a diet adequate in all nutrients (National Research Council, 1994) from d 2 to 7 posthatch. Following an overnight fast, chicks were weighed, wing-banded, and assigned to dietary treatments on d 8 posthatch in a randomized complete block design such that the average initial pen weights were not different among treatments. Eight replicate pens of 5 chicks received one of 7 treatment diets during a 14-d feeding study (d 8 to 22 posthatch). Battery pens (99.1 cm long, 33.7 cm wide, 26.7 cm high) provided 666.8 cm2 of floor space and access to 13.5 and 6.7 lineal cm/bird of feeder and water space, respectively, via hanging troughs in a room with adequate lighting, ventilation, and temperature control to meet agricultural standards. Chicks and feeders were weighed on d 8, 15, and 22 posthatch, and BW gain, feed intake, and G:F were calculated for each replicate pen of chicks. An Arg-deficient basal, formulated to contain 0.84% standardized ileal digestible (SID) Arg, was manufactured, composed primarily of corn, soybean meal, DDGS, and corn gluten meal (Table 1). Vitamin and mineral premixes, as well as crystalline AA, were incorporated into the basal to meet or exceed requirements for broiler chicks (National Research Council, 1994), with the exception of Arg (Table 2). Experimental treatment diets were subsequently produced by supplementing the basal with 2 levels of Arg (0 or 0.16% to provide 0.84 or 1.00% formulated SID Arg) and 3 levels of GAA (0, 0.06, or 0.12%) (CreAMINO®, GAA, 96% min, AlzChem AG, Trostberg, Germany) at the expense of corn to form a 2 × 3 factorial arrangement of dietary treatments (6 treatments). The unsupplemented basal diet (i.e., containing 0% supplementation of both Arg and GAA) served as the negative control (NC) based on established recommendations for dietary Arg (NRC, 1994). A seventh dietary treatment was produced by supplementing the basal diet with 0.32% Arg (1.16% SID Arg) to serve as the positive control (PC). All diets were fed in mash form in a single feeding phase that lasted from d 8 to 22 posthatch. Table 1. Basal diet formulation and nutrient composition. Ingredient, %    Value    Corn    57.57    Soybean Meal    10.03    DDGS    12.04    Corn Gluten Meal    11.54    Soy Oil    3.01    Salt    0.40    Limestone    1.50    Dicalcium Phosphate    1.71    Vitamin Premix1    0.20    Mineral Premix2    0.15    Choline Chloride    0.20    Titanium Dioxide    0.40    L-Lysine HCl    0.70    DL-Methionine    0.25    L-Isoleucine    0.06    L-Threonine    0.18    L-Tryptophan    0.05    Calculated Proximates       Crude Protein, %    21.3     Calcium, %    10.0     Phosphorus (total), %    6.8     Phosphorus (available), %    4.5     AMEN, kcal/kg    3148    Calculated Amino Acids, %  Total    SID3   Arg  0.96    0.84   Ile  0.86    0.76   Leu  2.53    2.31   Lys  1.24    1.14   Met  0.68    0.64   Met+Cys  1.04    0.92   Thr  0.87    0.73   Trp  0.23    0.19   Val  1.00    0.86  Ingredient, %    Value    Corn    57.57    Soybean Meal    10.03    DDGS    12.04    Corn Gluten Meal    11.54    Soy Oil    3.01    Salt    0.40    Limestone    1.50    Dicalcium Phosphate    1.71    Vitamin Premix1    0.20    Mineral Premix2    0.15    Choline Chloride    0.20    Titanium Dioxide    0.40    L-Lysine HCl    0.70    DL-Methionine    0.25    L-Isoleucine    0.06    L-Threonine    0.18    L-Tryptophan    0.05    Calculated Proximates       Crude Protein, %    21.3     Calcium, %    10.0     Phosphorus (total), %    6.8     Phosphorus (available), %    4.5     AMEN, kcal/kg    3148    Calculated Amino Acids, %  Total    SID3   Arg  0.96    0.84   Ile  0.86    0.76   Leu  2.53    2.31   Lys  1.24    1.14   Met  0.68    0.64   Met+Cys  1.04    0.92   Thr  0.87    0.73   Trp  0.23    0.19   Val  1.00    0.86  1Provided per kg of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 μg; DL-a- tocopheryl acetate, 11 IU; vitamin B12, 0.01 mg; riboflavin, 4.41 mg; D-Ca-pantothenate, 10 mg; niacin, 22 mg; menadione sodium bisulfite complex, 2.33 mg. 2Provided as milligrams per kg of diet: Mn, 75 from MnO; Fe, 75 from FeSO4 • 7H2O; Zn, 75 from ZnO; Cu, 5 from CuSO4 • 5H2O; I, 0.75 from ethylene diamine dihydroiodide; Se, 0.1 from Na2SeO3. 3Standardized ileal digestible AA composition calculated using data acquired from AMINODat® 4.0 (Evonik Industries AG, Hanau-Wolfgang, Germany). View Large Table 2. Analyzed composition of dietary treatments (as-is basis).   Dietary Treatment1  Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32  Nutrient GAA, %  0.002  0.06  0.12  0.00  0.06  0.12  0.003  Dry Matter, %  88.8  89.0  89.0  88.9  89.2  89.1  89.0  Crude Protein, %  21.4  21.9  20.7  21.8  22.2  21.4  23.0  Crude Fat, %  5.8  6.2  6.0  6.1  6.0  6.3  5.9  Crude Fiber, %  2.3  2.1  2.3  2.2  2.1  2.2  2.2  Ash, %  6.0  6.1  5.8  6.1  6.4  5.8  6.5  GAA, mg/kg4  <1.0  593  1160  <1.0  563  1130  <1.0  Creatine, mg/kg4  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  Folic Acid, mg/kg  0.94  0.87  0.97  0.85  1.00  1.08  0.92  Choline, mg/kg  1030  1160  1210  1110  1190  1080  1070  Betaine, mg/kg  6800  4900  7500  7300  6500  4000  7200  Total Amino Acids, %5  Essential   Arg  1.01  1.01  1.01  1.16  1.16  1.15  1.31   His  0.52  0.52  0.51  0.52  0.52  0.50  0.52   Ile  0.86  0.85  0.85  0.86  0.85  0.83  0.86   Leu  2.49  2.50  2.45  2.50  2.46  2.41  2.49   Lys  1.21  1.22  1.25  1.15  1.22  1.29  1.22   Met  0.68  0.64  0.66  0.66  0.65  0.68  0.66   Phe  1.10  1.11  1.09  1.11  1.09  1.07  1.10   Thr  0.87  0.91  0.85  0.90  0.90  0.88  0.89   Val  0.96  0.95  0.94  0.95  0.96  0.93  0.96  Non-Essential   Ala  1.45  1.45  1.41  1.45  1.42  1.39  1.43   Asp  1.55  1.56  1.56  1.57  1.57  1.53  1.56   Cys  0.38  0.38  0.37  0.38  0.38  0.37  0.38   Glu  3.87  3.89  3.83  3.90  3.85  3.77  3.87   Gly  0.76  0.76  0.76  0.76  0.76  0.75  0.76   Pro  1.57  1.55  1.53  1.57  1.55  1.51  1.56   Ser  1.02  1.03  1.00  1.03  1.01  1.00  1.01    Dietary Treatment1  Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32  Nutrient GAA, %  0.002  0.06  0.12  0.00  0.06  0.12  0.003  Dry Matter, %  88.8  89.0  89.0  88.9  89.2  89.1  89.0  Crude Protein, %  21.4  21.9  20.7  21.8  22.2  21.4  23.0  Crude Fat, %  5.8  6.2  6.0  6.1  6.0  6.3  5.9  Crude Fiber, %  2.3  2.1  2.3  2.2  2.1  2.2  2.2  Ash, %  6.0  6.1  5.8  6.1  6.4  5.8  6.5  GAA, mg/kg4  <1.0  593  1160  <1.0  563  1130  <1.0  Creatine, mg/kg4  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  Folic Acid, mg/kg  0.94  0.87  0.97  0.85  1.00  1.08  0.92  Choline, mg/kg  1030  1160  1210  1110  1190  1080  1070  Betaine, mg/kg  6800  4900  7500  7300  6500  4000  7200  Total Amino Acids, %5  Essential   Arg  1.01  1.01  1.01  1.16  1.16  1.15  1.31   His  0.52  0.52  0.51  0.52  0.52  0.50  0.52   Ile  0.86  0.85  0.85  0.86  0.85  0.83  0.86   Leu  2.49  2.50  2.45  2.50  2.46  2.41  2.49   Lys  1.21  1.22  1.25  1.15  1.22  1.29  1.22   Met  0.68  0.64  0.66  0.66  0.65  0.68  0.66   Phe  1.10  1.11  1.09  1.11  1.09  1.07  1.10   Thr  0.87  0.91  0.85  0.90  0.90  0.88  0.89   Val  0.96  0.95  0.94  0.95  0.96  0.93  0.96  Non-Essential   Ala  1.45  1.45  1.41  1.45  1.42  1.39  1.43   Asp  1.55  1.56  1.56  1.57  1.57  1.53  1.56   Cys  0.38  0.38  0.37  0.38  0.38  0.37  0.38   Glu  3.87  3.89  3.83  3.90  3.85  3.77  3.87   Gly  0.76  0.76  0.76  0.76  0.76  0.75  0.76   Pro  1.57  1.55  1.53  1.57  1.55  1.51  1.56   Ser  1.02  1.03  1.00  1.03  1.01  1.00  1.01  1The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. Abbreviations: GAA, guanidinoacetic acid. 2Negative control treatment. 3Positive control treatment. 4Analytical limit of detection was 1.0 mg/kg. 5Amino acids values were standardized to a dry matter content of 88%. View Large Sample Collection On d 22 posthatch, one non-fasted bird per pen was randomly chosen and euthanized via an intracardiac injection of 390 mg/mL sodium pentobarbital at 0.2 mL/kg BW to facilitate rapid collection of breast muscle tissue. A muscle biopsy sample, ranging from 1 to 5 g of wet tissue, was collected within 30 s of euthanasia and immediately immersed directly in liquid nitrogen until gas elaboration ceased. Time from euthanasia to flash-freezing of the muscle biopsy was no more than 60 s for any individual bird in order to prevent enzymatic PCr degradation. Snap-frozen muscle biopsy samples were then shattered using blunt force, with frozen muscle aliquots randomly dispensed into pre-cooled cryovials and placed back in liquid nitrogen until transferred to storage at −80°C. Once rapid muscle sample collection was complete, the bird was then passed to another station for additional collection of breast muscle, where the complete left pectoralis major muscle was removed by hand and placed into a pre-labeled storage bag. These samples were ultimately stored at −20°C until analysis of GAA and homocysteine as described below. Upon successful muscle collection, all remaining birds in each pen were euthanized by CO2 asphyxiation. Immediately after euthanasia, blood was separately collected from 2 of the remaining non-fasted birds per pen (randomly selected) via intracardiac puncture into evacuated tubes containing no anticoagulant, ethylenediaminetetraacetic acid (EDTA), or heparin to preserve serum, plasma, and whole-blood samples, respectively. Blood samples for plasma were stored on ice for no more than 1 h, and serum was allowed to clot at room temperature for no more than 2 h, before sample processing procedures began. Blood tubes for plasma and serum were centrifuged at 1,300 × g while being held at 4°C or 20°C, respectively, and all plasma and serum aliquots were subsequently stored at −80°C. Whole-blood samples were kept on ice until being stored at 4°C pending analysis. Following blood collection and processing steps, serum, plasma, and whole-blood samples from the 2 birds per pen were pooled into composite samples. Sample Analyses Diets were analyzed for dry matter, crude fat, crude fiber, and ash using standardized methods (AOAC International, 2006). Total nitrogen was determined using a Leco analyzer (TruMac N, Leco Corp., St. Joseph, MO) standardized with EDTA (method 990.03, AOAC International, 2006). Dietary AA concentrations were determined by ion-exchange chromatography with postcolumn derivatization with ninhydrin. Amino acids were oxidized with performic acid, which was neutralized with Na metabisulfite (Llames and Fontaine, 1994; Commission Directive, 1998). Amino acids were liberated from the protein by hydrolysis with 6 N HCl for 24 h at 110°C and quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. Tryptophan (Trp) was determined by high-performance liquid chromatography (HPLC) with fluorescence detection (extinction 280 nm, emission 356 nm), after alkaline hydrolysis with barium hydroxide octahydrate for 20 h at 110°C (Commission Directive, 2000). Tyrosine (Tyr) was not determined. Dietary folic acid, choline, and betaine were analyzed using validated methods (AOAC International, 2006) by an analytical laboratory (Eurofins Scientific Inc., Des Moines, IA). Dietary concentrations of GAA and Cr were quantified using fully validated procedures (Dobenecker and Braun, 2015) by an analytical laboratory (AlzChem AG, Trostberg, Germany). Muscle PCr, free Cr, and ATP concentrations were quantified using fully validated procedures (Harris et al., 1974; Swiss BioQuant, Reinach, Switzerland). In brief, muscle biopsy samples were freeze-dried, powdered, and following extraction with 1 mM EDTA in 0.5 M ice-cold perchloric acid (PCA) an aliquot of 25 μL of neutralized PCA extract (corresponding to 25 mg of dried muscle) was used for the simultaneous determination of ATP and PCr. For determination of free Cr, the neutralized extract was diluted with water (1:2 to 1:5 dilution). The analytical method was based on enzymatic determinations, which ultimately resulted in either reduction of NADP to NADPH (for ATP and PCr) or oxidation of NADH to NAD (for free Cr). For determination of ATP, 25 μL of the neutralized extract were mixed with 225 μL of TEA Buffer 1 (10 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 1 mM NADP, 0.04 mM ADP, 5 mM glucose, 4 μg/mL glucose-6-phosphate dehydrogenase in 100 mM TEA × HCl, pH 7.5) and the increase of absorbance at 340 nm was measured. Subsequently 10 μL creatine phosphokinase (5 mg/mL) was added to determine PCr based on the absorbance increase at 340 nm. For determination of Cr, 25 μL of the neutralized diluted extract were mixed with 225 μL of TEA Buffer 2 (10 mM magnesium acetate, 1 mM EDTA, 30 mM KCl, 1 mM phosphoenol pyruvate, 0.3 mM NADH, 2 mM ATP, 40 μg/mL pyruvate kinase, 20 μg/mL lactate dehydrogenase in 100 mM TEA × HCl, pH 8.5). After addition of 10 μL creatine phosphokinase (15 mg/mL in 0.5% NaHCO3, 0.05% BSA) the decrease of absorbance at 340 nm was measured. ATP, PCr and Cr contents were calculated based on the molar absorption coefficient for NADH at 340 nm (6.22 mM–1 × cm–1). Muscle glycogen concentrations were quantified using fully validated procedures (Swiss BioQuant, Reinach, Switzerland). In brief, 10 mg of freeze-dried muscle was hydrolyzed in 0.5 mL of 1 M HCl for 2 h at approximately 99°C to convert glycogen to glucose. The hydrolysate was then neutralized by the addition of 0.15 mL (per 0.5 mL hydrolysate) of 0.1 M imidazol maintained at pH 7.0 using 0.1 M NaOH. Following centrifugation at 13,000 × g for 5 min, the supernatant was diluted 1:20 with water. Subsequently, the supernatants were subjected to a glucose analysis based on the total hydrolysis of glycogen to glucose by enzymatic determination using a commercially available glucose oxidase assay (GAGO-20; Sigma-Aldrich, St. Louis, MO). Homocysteine was determined in wet muscle tissue by ion chromatography (Dionex DX500, with gradient pump and fluorescence detector) using a fully validated procedure (LiChrospher 100 RP-18 column, 4.6 × 250 mm, 5 μm; column temperature 30°C, flow rate set at 1.0 mL min–1). In brief, 100 mg of the minced wet tissue sample was weighed into a reaction flask, 400 μL phosphate salt buffer (pH 7.4) was added, and the suspension homogenized thoroughly using a rotor-stator mixer. An aliquot of the homogenate was reacted with tri-n-butylphosphine solution at 4°C, and after addition of 0.6 M perchloric acid, the suspension was centrifuged. The supernatant was subsequently mixed with borate buffer (pH 10.5) and SBDF reagent (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, 1 g/L in borate buffer), and the resulting solution was heated for 60 min at 60°C and then centrifuged for 10 min at 20,000 rpm after cooling to room temperature. Finally, the supernatant was used for quantification of homocysteine, using a mobile phased produced by dissolving 5.86 g sodium acetate in water in a 1-L volumetric flask, and subsequently adding 1.72 g glacial acetic acid and 20 mL methanol prior to filling the volumetric flask to volume with water. Serum [AA, Cr, creatinine (Crn), GAA] and plasma samples (homocysteine) were analyzed using standardized procedures (Baylor University, Houston, TX). Whole blood was submitted to the University of Illinois Urbana-Champaign Veterinary Diagnostic Laboratory for analysis of hematological and clinical pathology parameters. Blood biochemistry was assayed using an automated spectrophotometric method on a Hitachi 917 analyzer (Roche, Indianapolis, IN), while hematological parameters were assessed using a combination of automated and manual procedures. Statistical Analysis Data from all diets except the PC were analyzed as a 2-way analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Inst., Cary, NC); dietary supplemental Arg and GAA concentrations were independent variables in the statistical model. Whereas birds were allotted based on a randomized complete block design, the fixed effect of block was insignificant in all cases and was therefore removed from the statistical model. When interactive effects were noted, means separation was conducted using a Tukey's adjustment. In addition to the 2-way ANOVA, each dietary treatment was compared to the PC diet using a 2-tailed Dunnett's test. Overall treatment effects with a probability of P < 0.05 were accepted as statistically significant. RESULTS Overall, formulation objectives were achieved in terms of creating an Arg-deficient basal diet, and graded supplementation of Arg and GAA was realized in the final diets (Table 2). Mortality was only 1.43% and was not affected by dietary treatment. Growth Performance Feed intake was not affected by any of the dietary treatments and no interactive effects were noted for any performance parameter in this study. Body weight gain d 15 to 22 and d 8 to 22 posthatch were increased (P < 0.05) due to the main effect of Arg supplementation, with the NC diet (0% supplemental Arg and GAA) exhibiting reduced (P < 0.05) BW gain compared with the PC diet for d 15 to 22 (Table 3). No main effects of GAA supplementation was observed for BW gain regardless of feeding period, though a trend for GAA to increase BW gain at d 15 to 22 was observed (P = 0.094). Feed efficiency (i.e., G:F) was independently increased (P < 0.05) due to supplementation of either 0.16% Arg or graded dietary GAA (i.e., main effects of both Arg and GAA) from d 15 to 22 and d 8 to 22 posthatch to reach similar performance as elicited by the PC diet. Table 3. Growth performance of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Body weight, g   d8  99.3  99.4  99.4  99.3  99.5  99.5  99.3  2.83  1.00  1.00  1.00   d15  299.8  285.4  286.3  293.6  299.7  300.4  301.1  10.02  0.63  0.90  0.51   d22  627.9  626.2  641.0  642.0  656.3  662.2  668.5  16.37  0.076  0.59  0.89  BW gain, g/chick   d 8 to 15  200.5  186.0  186.9  194.3  200.2  200.9  201.8  7.52  0.44  0.83  0.31   d 15 to 22  328.1*  340.9  354.7  348.4  356.6  361.8  367.4  8.99  0.008  0.094  0.76   d 8 to 22  528.6  526.8  541.6  542.7  556.8  562.7  569.2  14.39  0.037  0.52  0.86  Feed intake, g/chick   d 8 to 15  301.4  278.9  278.6  282.1  295.1  283.1  286.9  9.37  0.85  0.47  0.13   d 15 to 22  514.0  516.3  512.4  509.4  518.7  503.3  523.6  13.60  0.77  0.75  0.90   d 8 to 22  815.6  797.7  791.0  791.5  813.8  791.3  810.8  20.98  0.98  0.73  0.59  G:F, g/kg   d 8 to 15  663.8  666.8  668.9  687.0  680.9  711.4  697.4  17.94  0.100  0.57  0.69   d 15 to 22  637.9*  661.4  693.0  683.9  690.5  718.1  701.9  15.43  0.003  0.011  0.75   d 8 to 22  647.3*  661.5  684.3  685.1  686.9  710.9  700.5  13.63  0.004  0.047  0.87    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Body weight, g   d8  99.3  99.4  99.4  99.3  99.5  99.5  99.3  2.83  1.00  1.00  1.00   d15  299.8  285.4  286.3  293.6  299.7  300.4  301.1  10.02  0.63  0.90  0.51   d22  627.9  626.2  641.0  642.0  656.3  662.2  668.5  16.37  0.076  0.59  0.89  BW gain, g/chick   d 8 to 15  200.5  186.0  186.9  194.3  200.2  200.9  201.8  7.52  0.44  0.83  0.31   d 15 to 22  328.1*  340.9  354.7  348.4  356.6  361.8  367.4  8.99  0.008  0.094  0.76   d 8 to 22  528.6  526.8  541.6  542.7  556.8  562.7  569.2  14.39  0.037  0.52  0.86  Feed intake, g/chick   d 8 to 15  301.4  278.9  278.6  282.1  295.1  283.1  286.9  9.37  0.85  0.47  0.13   d 15 to 22  514.0  516.3  512.4  509.4  518.7  503.3  523.6  13.60  0.77  0.75  0.90   d 8 to 22  815.6  797.7  791.0  791.5  813.8  791.3  810.8  20.98  0.98  0.73  0.59  G:F, g/kg   d 8 to 15  663.8  666.8  668.9  687.0  680.9  711.4  697.4  17.94  0.100  0.57  0.69   d 15 to 22  637.9*  661.4  693.0  683.9  690.5  718.1  701.9  15.43  0.003  0.011  0.75   d 8 to 22  647.3*  661.5  684.3  685.1  686.9  710.9  700.5  13.63  0.004  0.047  0.87  *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. View Large Breast Muscle Analysis Tissue metabolite concentrations were expressed both as absolute concentrations as well as relative concentrations (Table 4), normalized to ATP in order to standardize for lean muscle tissue as suggested by Harris et al. (1992). Birds fed the NC diet exhibited lower (P < 0.05) absolute and relative concentrations of muscle PCr, as well as absolute total Cr, when compared to birds fed the PC diet. Muscle absolute and relative PCr concentrations independently increased (P < 0.05) due to dietary addition of either 0.16% Arg or graded GAA supplementation, but no interactive effects were noted. Highest metabolite concentrations were obtained from supplementation of the basal diet with both 0.16% Arg and 0.12% GAA, which resulted in absolute concentrations of muscle PCr being increased (P < 0.05) by 189% and 46% compared with the NC and PC diets, respectively. Similarly, the PCr: ATP ratio was increased by 227% and 72% compared with the NC and PC diets. Graded GAA supplementation increased (P < 0.05) muscle total Cr concentrations, but the effect was more pronounced in diets containing supplemental Arg (interaction, P < 0.05). The addition of 0.16% Arg and 0.12% GAA resulted in total Cr concentrations that were increased (P < 0.05) by 26% compared with the PC diet. Muscle glycogen was independently increased (main effects; P < 0.05) by supplementation of either Arg or GAA. Muscle GAA was measured but not reported due to most samples containing GAA concentrations below the detection limit (<5 mg/kg). Homocysteine concentrations in wet muscle were not affected by dietary treatment. Table 4. Muscle analyses of energy-related metabolites of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Absolute concentrations   ATP, mmol/kg DW  37.8  38.0  41.0  38.1  40.8  34.1  40.1  2.36  0.62  0.71  0.11   PCr, mmol/kg DW  37.6*  56.1  91.0  61.7  82.4  108.7*  74.4  8.14  0.001  <0.001  0.86   Free Cr, mmol/kg DW  68.9  56.3  59.5  54.7  76.5  67.3  64.7  7.81  0.74  0.84  0.094   Total Cr, mmol/kg DW7  106.6a*  112.4a*  150.6b  116.5a  158.9b  176.0b*  139.2  7.13  <0.001  <0.001  0.044   Glycogen, mmol/kg DW  171.5  221.9  260.8  239.8  266.1  310.1  265.0  26.75  0.008  0.010  0.88  Relative concentrations   PCr:ATP ratio  0.99*  1.54  2.20  1.68  2.04  3.24*  1.88  0.24  <0.001  <0.001  0.52   Free Cr:ATP ratio  1.92  1.58  1.52  1.45  1.95  2.03  1.66  0.27  0.82  0.94  0.15   Total Cr:ATP ratio  2.91  3.11  3.73  3.12  4.00  5.27*  3.55  0.30  0.001  <0.001  0.091   PCr/total Cr, %  34.5  51.0  60.5  52.2  51.9  61.7  53.7  5.40  0.11  0.007  0.22    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Absolute concentrations   ATP, mmol/kg DW  37.8  38.0  41.0  38.1  40.8  34.1  40.1  2.36  0.62  0.71  0.11   PCr, mmol/kg DW  37.6*  56.1  91.0  61.7  82.4  108.7*  74.4  8.14  0.001  <0.001  0.86   Free Cr, mmol/kg DW  68.9  56.3  59.5  54.7  76.5  67.3  64.7  7.81  0.74  0.84  0.094   Total Cr, mmol/kg DW7  106.6a*  112.4a*  150.6b  116.5a  158.9b  176.0b*  139.2  7.13  <0.001  <0.001  0.044   Glycogen, mmol/kg DW  171.5  221.9  260.8  239.8  266.1  310.1  265.0  26.75  0.008  0.010  0.88  Relative concentrations   PCr:ATP ratio  0.99*  1.54  2.20  1.68  2.04  3.24*  1.88  0.24  <0.001  <0.001  0.52   Free Cr:ATP ratio  1.92  1.58  1.52  1.45  1.95  2.03  1.66  0.27  0.82  0.94  0.15   Total Cr:ATP ratio  2.91  3.11  3.73  3.12  4.00  5.27*  3.55  0.30  0.001  <0.001  0.091   PCr/total Cr, %  34.5  51.0  60.5  52.2  51.9  61.7  53.7  5.40  0.11  0.007  0.22  a-bMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: ATP = adenosine triphosphate, Cr = creatine, DW = dry weight, GAA = guanidinoacetic acid, PCr = phosphocreatine, WW = wet weight. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. 7Calculated as PCr plus free Cr. View Large Blood Analysis Serum Arg concentrations were unchanged by graded GAA in diets containing 0% added Arg, but increased 38% due to addition of 0.12% GAA in diets containing 0.16% added Arg (interaction, P < 0.05; Table 5). Interactive effects (P < 0.05) were also observed for histidine (His), isoleucine (Ile), lysine (Lys), phenylalanine (Phe), and valine (Val), which decreased an average of 34 and 7% due to addition of 0.12% GAA when included in diets containing 0.0 or 0.16% added Arg, respectively. Glutamine (Gln) was the only non-essential AA that exhibited an interaction (P < 0.05), with all other non-essential AA (except asparagine [Asn]) decreasing (P < 0.05) due to Arg supplementation. Additionally, alanine (Ala) decreased (P < 0.05) due to graded addition of GAA, regardless of dietary Arg concentration. Table 5. Serum amino acid and metabolite concentrations (μM) of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Essential   Arginine  155.8a,b,*  136.9a*  143.8a*  199.1b,c,*  244.6c,d,*  274.0d*  364.9  15.35  <0.001  0.13  0.019   Histidine  127.0c*  109.1b,c  79.6a,b  80.9a,b  74.1a,b  73.4a  84.7  8.03  <0.001  0.005  0.046   Isoleucine  118.4b*  89.4a  82.9a  79.6a  82.9a  76.8a  77.3  4.26  <0.001  0.001  0.001   Leucine  434.3*  354.4  334.5  370.3  337.4  318.4  341.1  16.92  0.002  0.001  0.28   Lysine  579.0c*  470.6b,c*  272.5a  297.3a,b  237.8a  262.5a  274.3  43.58  <0.001  0.001  0.007   Methionine  196.4  181.3  179.6  177.0  158.8  162.0  161.6  10.64  0.017  0.22  0.97   Phenylalanine  217.5  184.1  184.4  188.3  198.8  186.1  172.9  8.67  0.021  0.13  0.042   Threonine  2093*  1839*  1333  1410  1243  1175  1254  113.18  <0.001  0.001  0.054   Valine  245.1b*  173.3a  160.9a  164.3a  158.3a  148.6a  150.8  8.33  <0.001  <0.001  0.001  Non-Essential   Alanine  1328*  1148  1077  1055  991.1  950.1  927.3  71.08  0.001  0.047  0.56   Asparagine  20.9  43.5  60.3  41.8  59.6  35.0  44.6  21.79  0.86  0.62  0.51   Aspartic acid  221.9*  220.6*  185.6  176.1  162.6  179.1  157.1  13.32  0.001  0.46  0.14   Cysteine  85.6*  80.9  75.1  75.0  75.8  72.5  72.9  3.34  0.021  0.15  0.48   Glutamine  1,243b  1,514c*  1,180b  1,095a,b  1,070a,b  882.6a*  1095  54.66  <0.001  <0.001  0.033   Glutamic acid  266.0*  250.5  254.4  224.9  229.9  254.4  224.5  9.43  0.009  0.32  0.10   Glycine  731.3*  696.3*  632.0*  520.5  521.8  517.0  476  38.27  <0.001  0.40  0.46   Proline  832.8  883.9  886.4  756.3  748.0  745.3  768.5  52.37  0.027  0.90  0.79   Serine  936.5*  918.0*  800.0  721.9  680.5  652.3  715.0  48.08  <0.001  0.098  0.63   Tyrosine  409.6  422.0  365.0  329.9  363.6  337.5  326.9  26.80  0.024  0.31  0.62  Other Metabolites   Alpha-aminobutyric acid  71.9c,*  34.8a,*  50.5b,*  42.5a,b,*  37.5a,b,*  36.4a,b,*  20.4  3.31  <0.001  <0.001  <0.001   Citrulline  2.75a,*  9.0b  15.9c,*  15.8c,*  14.9c,*  16.8c,*  9.5  1.07  <0.001  <0.001  <0.001   Ornithine  11.0*  7.0*  7.38*  20.1*  17.3*  17.6*  28.0  1.78  <0.001  0.12  0.94   1-Methylhistidine  38.1*  30.4  24.6  26.6  27.4  22.8  20.5  2.87  0.001  0.014  0.20   3-Methylhistidine  16.3c,*  12.6b  7.13a  9.25a,b  7.25a  8.88a,b  11.0  1.05  0.001  0.001  0.001   Phosphoserine  20.6a  19.0a  70.8b,*  75.9b,*  75.5b,*  68.9b,*  20.6  3.47  <0.001  <0.001  <0.001   Phosphoethanolamine  2.75a,b  2.25a  4.13a,b,*  14.9c,*  4.38b,*  4.50b,*  1.63  0.48  <0.001  <0.001  <0.001   Taurine  362.9  297.8  340.5  381  288.8  311.8  286.9  29.82  0.074  0.038  0.73    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Essential   Arginine  155.8a,b,*  136.9a*  143.8a*  199.1b,c,*  244.6c,d,*  274.0d*  364.9  15.35  <0.001  0.13  0.019   Histidine  127.0c*  109.1b,c  79.6a,b  80.9a,b  74.1a,b  73.4a  84.7  8.03  <0.001  0.005  0.046   Isoleucine  118.4b*  89.4a  82.9a  79.6a  82.9a  76.8a  77.3  4.26  <0.001  0.001  0.001   Leucine  434.3*  354.4  334.5  370.3  337.4  318.4  341.1  16.92  0.002  0.001  0.28   Lysine  579.0c*  470.6b,c*  272.5a  297.3a,b  237.8a  262.5a  274.3  43.58  <0.001  0.001  0.007   Methionine  196.4  181.3  179.6  177.0  158.8  162.0  161.6  10.64  0.017  0.22  0.97   Phenylalanine  217.5  184.1  184.4  188.3  198.8  186.1  172.9  8.67  0.021  0.13  0.042   Threonine  2093*  1839*  1333  1410  1243  1175  1254  113.18  <0.001  0.001  0.054   Valine  245.1b*  173.3a  160.9a  164.3a  158.3a  148.6a  150.8  8.33  <0.001  <0.001  0.001  Non-Essential   Alanine  1328*  1148  1077  1055  991.1  950.1  927.3  71.08  0.001  0.047  0.56   Asparagine  20.9  43.5  60.3  41.8  59.6  35.0  44.6  21.79  0.86  0.62  0.51   Aspartic acid  221.9*  220.6*  185.6  176.1  162.6  179.1  157.1  13.32  0.001  0.46  0.14   Cysteine  85.6*  80.9  75.1  75.0  75.8  72.5  72.9  3.34  0.021  0.15  0.48   Glutamine  1,243b  1,514c*  1,180b  1,095a,b  1,070a,b  882.6a*  1095  54.66  <0.001  <0.001  0.033   Glutamic acid  266.0*  250.5  254.4  224.9  229.9  254.4  224.5  9.43  0.009  0.32  0.10   Glycine  731.3*  696.3*  632.0*  520.5  521.8  517.0  476  38.27  <0.001  0.40  0.46   Proline  832.8  883.9  886.4  756.3  748.0  745.3  768.5  52.37  0.027  0.90  0.79   Serine  936.5*  918.0*  800.0  721.9  680.5  652.3  715.0  48.08  <0.001  0.098  0.63   Tyrosine  409.6  422.0  365.0  329.9  363.6  337.5  326.9  26.80  0.024  0.31  0.62  Other Metabolites   Alpha-aminobutyric acid  71.9c,*  34.8a,*  50.5b,*  42.5a,b,*  37.5a,b,*  36.4a,b,*  20.4  3.31  <0.001  <0.001  <0.001   Citrulline  2.75a,*  9.0b  15.9c,*  15.8c,*  14.9c,*  16.8c,*  9.5  1.07  <0.001  <0.001  <0.001   Ornithine  11.0*  7.0*  7.38*  20.1*  17.3*  17.6*  28.0  1.78  <0.001  0.12  0.94   1-Methylhistidine  38.1*  30.4  24.6  26.6  27.4  22.8  20.5  2.87  0.001  0.014  0.20   3-Methylhistidine  16.3c,*  12.6b  7.13a  9.25a,b  7.25a  8.88a,b  11.0  1.05  0.001  0.001  0.001   Phosphoserine  20.6a  19.0a  70.8b,*  75.9b,*  75.5b,*  68.9b,*  20.6  3.47  <0.001  <0.001  <0.001   Phosphoethanolamine  2.75a,b  2.25a  4.13a,b,*  14.9c,*  4.38b,*  4.50b,*  1.63  0.48  <0.001  <0.001  <0.001   Taurine  362.9  297.8  340.5  381  288.8  311.8  286.9  29.82  0.074  0.038  0.73  a-dMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. View Large Serum concentrations of citrulline, phosphoserine, and phosphoethanolamine increased due to GAA addition to Arg-unsupplemented diets, but were unchanged when GAA was added to diets containing supplemental Arg (interaction, P < 0.05). Opposite interactive effects (P < 0.05) were observed for alpha-aminobutyric acid and 3-methylhistidine, which decreased due to GAA addition to Arg-unsupplemented diets, but were unchanged when GAA was added to diets containing supplemental Arg. Serum ornithine increased (P < 0.05) 117% due to Arg supplementation (i.e., PC vs. NC), and a main effect (P < 0.05) of GAA supplementation caused a decrease in taurine concentrations. Finally, serum 1-methylhistidine decreased (P < 0.05) by 18 and 27% due to supplementation with either 0.16% Arg or 0.12% GAA, respectively. In general, the NC diet elicited lower serum Cr concentrations as compared with birds fed the PC diet, while serum GAA and plasma homocysteine were not different between NC- and PC-fed birds. Graded GAA supplementation increased (P < 0.05) serum GAA concentrations, but the effect was more pronounced in diets containing 0.16% supplemental Arg (interaction, P < 0.05; Table 6). Moreover, addition of either 0.06 or 0.12% GAA increased (P < 0.05) serum GAA by at least 323% when compared with the PC diet. Serum Cr concentrations independently increased (P < 0.05) due to dietary addition of either Arg or GAA, and no interactive effect was observed. Serum Crn was analyzed to be below detection limits (<4.4 μM) in all samples, and therefore data are not available, and plasma homocysteine was not affected by dietary treatment. Table 6. Blood and muscle concentrations of creatine-related metabolites from chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Plasma   Homocysteine, μM  155.4  151.6  150.5  178.2  170.5  157.7  134.4  13.42  0.057  0.64  0.83   Uric acid, μM  8.5a*  8.3a*  6.5a,b  6.2a,b  5.7b  6.9a,b  5.9  0.59  0.002  0.52  0.028  Serum   Creatine, μM  14.2*  22.3  37.1  19.8  26.0  43.2*  28.3  3.31  0.006  <0.001  0.93   Guanidinoacetic acid, μM  0.39d  3.85c*  9.84b*  0.48d  4.91c*  13.95a*  0.91  0.54  <0.001  <0.001  0.001  Muscle7   Homocysteine, μmol/kg8  75.3  114.3  82.7  108.6  78.3  70.9  119.0  39.96  0.28  0.36  0.054    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Plasma   Homocysteine, μM  155.4  151.6  150.5  178.2  170.5  157.7  134.4  13.42  0.057  0.64  0.83   Uric acid, μM  8.5a*  8.3a*  6.5a,b  6.2a,b  5.7b  6.9a,b  5.9  0.59  0.002  0.52  0.028  Serum   Creatine, μM  14.2*  22.3  37.1  19.8  26.0  43.2*  28.3  3.31  0.006  <0.001  0.93   Guanidinoacetic acid, μM  0.39d  3.85c*  9.84b*  0.48d  4.91c*  13.95a*  0.91  0.54  <0.001  <0.001  0.001  Muscle7   Homocysteine, μmol/kg8  75.3  114.3  82.7  108.6  78.3  70.9  119.0  39.96  0.28  0.36  0.054  a-dMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. 7Muscle GAA was undetectable in most samples, and is therefore not reported. 8Units are expressed relative to wet tissue weight. View Large Clinical chemistry and hematological outcomes were largely unaffected by dietary treatment (Table 7). An interaction (P < 0.05) was observed for glucose, with 0.12% added GAA increasing glucose concentrations by 2.7% in Arg-unsupplemented diets and decreasing glucose concentrations by 4.1% in Arg-supplemented diets. Plasma phosphorus concentrations and glutamic acid (Glu) dehydrogenase activity were both decreased (P < 0.05) due to Arg supplementation, and Glu dehydrogenase activity was also decreased (P < 0.05) due to GAA supplementation. In terms of hematological responses, blood protein concentrations decreased (P < 0.05) due to supplementation of either Arg or GAA (Table 8). As a proportion of total blood leukocytes, heterophils decreased (P < 0.05), while lymphocytes increased (P < 0.05), due to graded GAA supplementation; no other hematological outcomes were affected by dietary treatment. Table 7. Blood clinical pathology outcomes of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Albumin, g/dL  1.10  1.13  1.14  1.1  1.00  1.08  1.05  0.05  0.24  0.56  0.43  Glucose, mg/dL  232.1a,b  244.6b  238.3a,b  241.5a,b  220.4a  231.5a,b  236.8  5.36  0.27  0.73  0.011  Asp aminotransferase, U/L  136.75  148.25  143.88  135  131.75  146.38  145  7.37  0.42  0.46  0.41  Phosphorus, mg/dL  7.60  8.39  7.98  7.53  7.20  7.39  7.68  0.27  0.027  0.70  0.14  Calcium, mg/dL  10.69  10.85  10.68  10.61  10.28  10.85  10.45  0.21  0.50  0.65  0.21  Glu dehydrogenase, U/L  6.78  4.76  5.40  4.95  3.33  3.69  4.81  0.66  0.007  0.025  0.96  Creatine kinase, U/L  2106  1908  2586  2672  2133  2379  2178  304.0  0.62  0.28  0.42    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Albumin, g/dL  1.10  1.13  1.14  1.1  1.00  1.08  1.05  0.05  0.24  0.56  0.43  Glucose, mg/dL  232.1a,b  244.6b  238.3a,b  241.5a,b  220.4a  231.5a,b  236.8  5.36  0.27  0.73  0.011  Asp aminotransferase, U/L  136.75  148.25  143.88  135  131.75  146.38  145  7.37  0.42  0.46  0.41  Phosphorus, mg/dL  7.60  8.39  7.98  7.53  7.20  7.39  7.68  0.27  0.027  0.70  0.14  Calcium, mg/dL  10.69  10.85  10.68  10.61  10.28  10.85  10.45  0.21  0.50  0.65  0.21  Glu dehydrogenase, U/L  6.78  4.76  5.40  4.95  3.33  3.69  4.81  0.66  0.007  0.025  0.96  Creatine kinase, U/L  2106  1908  2586  2672  2133  2379  2178  304.0  0.62  0.28  0.42  a,bMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. View Large Table 8. Hematological outcomes of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-Value6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Packed cell volume, %  28.7  28.2  29.1  28.3  28.5  28.9  28.5  0.86  0.99  0.68  0.90  Protein, g/dL  3.36  3.03  3.15  3.18  2.86  2.99  3.03  0.96  0.009  0.003  1.00  Total leukocytes, 103/μL  10.65  12.88  11.08  9.86  9.99  13.97  9.89  1.95  0.97  0.46  0.27  Differential cell proportions, %7   Heterophils  61.0  40.0  35.3  51.0  44.0  37.7  44.0  5.63  0.17  0.001  0.33   Lymphocytes  28.4  44.5  52.2  35.9  40.9  49.6  43.9  6.37  0.27  0.008  0.57   Monocytes  3.67  4.60  2.75  5.13  5.25  5.40  4.71  1.86  0.50  0.88  0.84   Eosinophils  2.80  2.50  1.57  2.57  2.29  2.29  2.25  0.67  0.83  0.36  0.60   Basophils  5.43  8.00  8.38  6.67  7.88  6.57  6.00  1.73  0.84  0.26  0.57    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-Value6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Packed cell volume, %  28.7  28.2  29.1  28.3  28.5  28.9  28.5  0.86  0.99  0.68  0.90  Protein, g/dL  3.36  3.03  3.15  3.18  2.86  2.99  3.03  0.96  0.009  0.003  1.00  Total leukocytes, 103/μL  10.65  12.88  11.08  9.86  9.99  13.97  9.89  1.95  0.97  0.46  0.27  Differential cell proportions, %7   Heterophils  61.0  40.0  35.3  51.0  44.0  37.7  44.0  5.63  0.17  0.001  0.33   Lymphocytes  28.4  44.5  52.2  35.9  40.9  49.6  43.9  6.37  0.27  0.008  0.57   Monocytes  3.67  4.60  2.75  5.13  5.25  5.40  4.71  1.86  0.50  0.88  0.84   Eosinophils  2.80  2.50  1.57  2.57  2.29  2.29  2.25  0.67  0.83  0.36  0.60   Basophils  5.43  8.00  8.38  6.67  7.88  6.57  6.00  1.73  0.84  0.26  0.57  1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. 7Differential cell counts expressed as a proportion of total leukocytes detected. View Large DISCUSSION This study was designed to test the efficacy of GAA for restoring growth performance and muscle phosphagen status in fast-growing broiler chicks fed an Arg-deficient diet based on practical ingredients. Combined with a general lack of effects on hematological and clinical chemistry outcomes, this research provides clear and direct evidence that: 1) Arg deficiency is detrimental to growth performance and produces disruptions in blood and muscle energy metabolites, i.e., Cr and PCr profiles, as well as glycogen levels, and 2) supplementation with Arg and/or GAA ameliorate these effects, though to varying degrees depending on the dietary nutrient. Of particular interest were the findings that Arg and GAA supplementation restored BW gain and G:F parameters elicited by Arg deficiency, as well as robust increases in muscle PCr, both on an absolute basis and relative to the muscle ATP concentration as well as muscle glycogen levels. Improvements in growth performance when GAA was added to Arg-deficient diets are in agreement with previous research conducted by Savage and O’Dell (1960) and Dilger et al. (2013). Whereas there was no BW gain response due to GAA supplementation, this was also in agreement with previous research (Dilger et al., 2013). It should be noted that broilers received a standard starter diet (corn-soybean meal-based) that was adequate in all nutrients, including Arg, prior to initiation of the experimental phase. Acknowledging that there is no ability to store AA, it is possible that pre-testing on an Arg-deficient diet may have altered responses to supplemental Arg and GAA during the 2-week growth period used in our study. Improvements in G:F likely occurred because Arg was spared from serving as a precursor for Cr synthesis (Almquist et al., 1941) and was therefore available for alternative functions throughout the body (e.g., lean tissue accretion); a theory that is in agreement with the research conducted by Edwards et al. (1958). In general, GAA was able to improve, and in some instances restore, growth performance of birds receiving Arg-deficient diets containing practical ingredients common to the U.S. poultry industry. In our study, dietary Arg deficiency decreased concentrations of muscle total Cr, and consequently muscle PCr, compared with the PC diet. Because PCr is used to maintain muscle energy homeostasis (Walker, 1979), the decrease in PCr means that muscles in Arg-deficient birds were less able to maintain energy homeostasis, hence they may have been less tolerable to ATP-consuming processes under stressful conditions (e.g., oxidative stress, disease challenge). Regardless of Arg supplementation level, GAA increased muscle PCr such that they were equal to or greater than those elicited by the PC diet. This finding indicates that GAA improved energy homeostasis in muscles cells when supplemented in an Arg-deficient diet. Relative to the NC diet, total Cr concentrations in tissues were increased due to 0.12% GAA supplementation by 64% or 59% in diets formulated to contain 0.84% or 1.00% SID Arg, respectively. Supplementation of 0.06% GAA elicited significant improvements, primarily when added to diets containing 1.00% SID Arg. These results corroborate recent observations by Michiels et al. (2012), where improvements in Cr and PCr/ATP concentrations in breast meat were reported as a result of graded GAA supplementation of Arg-adequate diets. Because maintenance of PCr is based on Cr being phosphorylated to PCr and exported to sites of ATP usage (Brosnan and Brosnan, 2010; Guimarães-Ferreira, 2014), the increased total Cr concentration may indicate an increased capacity for PCr synthesis. This is confirmed by both the absolute concentration and relative ratio (to ATP) of PCr and total Cr, which were both elevated with GAA supplementation, indicating in both cases that GAA may increase the ability of cells to regenerate ATP more effectively. Because of these increases in muscle metabolite concentrations, and the fact that serum GAA concentrations were responsive to dietary GAA supplementation, we conclude that dietary GAA was successfully absorbed and metabolized to synthesize Cr with efficacy greater than that of dietary Arg, which indicates that GAA supplementation will ultimately spare dietary Arg (Wyss and Kaddurah-Daouk, 2000). Considering the rate-limiting enzyme for Cr synthesis, AGAT, is almost exclusively inhibited by Cr (Walker, 1979), increased production of Cr via dietary GAA supplementation would likely exert Arg-sparing effects by downregulating expression and/or activity of the AGAT enzyme, thereby permitting more dietary Arg to be used for purposes other than Cr synthesis. Arg deficiency was visible from reduced serum concentrations in Arg and elevated serum concentrations of most other essential and non-essential AA. Supplementation of GAA reduced serum AA concentrations in birds, with more drastic decreases observed in diets containing 0.84% SID Arg. This indicates that as the Arg deficiency was alleviated, essential AA concentrations in plasma decreased. Zimmerman and Scott (1965) reported a similar trend in BW gain and plasma AA concentrations as dietary lysine approached the requirement. The improvement in BW gain and decrease in serum AA may be explained in the context of the ideal protein concept, which is based on the premise that optimal performance is limited by availability of the scarcest resource. In these diets, Arg is the most-limiting AA, and as the body has no true AA storage depots, any excess AA remaining after attaining maximal growth are transported via the blood to the liver for deamination and subsequent oxidation. Therefore, GAA added to diets formulated to contain either 0.84% or 1.00% SID Arg may have spared Arg by diminishing the need for Arg to serve as a precursor for Cr synthesis, therefore making more Arg available for incorporation into lean tissue along with excess AA, to ultimately decrease serum AA concentrations. In addition to decreased growth in general, evidence from blood chemistry and hematological outcomes indicate that protein degradation may have occurred in Arg-deficient birds. Serum 3-methylhistidine decreased with dietary supplementation of GAA to Arg-unsupplemented diets, but was unchanged when GAA was added to Arg-supplemented diets. Supplementation of GAA also decreased heterophils as the largest proportion of total leukocytes, while dietary Arg concentration had no effect. Heterophil proportions were reduced with GAA supplementation an average of 35% when birds were supplemented with 0.12% GAA compared with 0.0% GAA. There was also a reciprocal change in lymphocyte percentages, the normal circulating leukocyte (Weiss et al., 2010), as heterophil percentage decreased. The combination of decreased 3-methylhistidine concentrations, total protein and heterophil percentages in birds with increased GAA supplementation indicates that Arg deficiency may have caused minor changes in whole-body protein degradation. Although 3-methylhistidine is an indicator of skeletal protein degradation (Young and Munro, 1978), it is typically related to body weight loss. However, when combined with the high proportion of white blood cells as heterophils, a sign of muscle damage (Weiss et al., 2010), this may suggest greater whole-body protein breakdown occurred in Arg-deficient birds. In this context, GAA supplementation may be seen as having reduced metabolic stress (i.e., protein degradation) caused by dietary Arg deficiency. Alleviation of an Arg deficiency is associated with changes in Arg-related metabolites. Serum ornithine (Orn) concentrations were reduced in birds fed the NC vs. PC diet and increased with Arg supplementation, but GAA supplementation had no effect on this circulating metabolite. The production of Orn may increase with Arg supplementation due to the increased production of urea by avian arginase (Tamir and Ratner, 1963a; Fernandes and Murakami, 2010). Increased serum Orn concentrations may also indicate that more GAA and Orn were formed from Arg and Gly due to Arg supplementation (Walker, 1979). Supplementation of GAA decreased Orn concentrations by at least 33 and 12% in diets formulated to contain 0.84 or 1.00% SID Arg with 0.0% GAA, respectively. This was expected because supplementation of GAA spares dietary Arg to the greatest extent in Arg-deficient diets. Serum citrulline levels were also lower in birds fed NC vs. PC diets, but were increased with supplementation of GAA, although to a much lower effect when dietary Arg was closer to adequate concentrations. This could be due to increased nitric oxide production, which also results in citrulline production (Wu and Morris, 1998), but also a decreased conversion of citrulline to Arg (Tamir and Ratner, 1963b) in chicks under less-deficient conditions. As serum citrulline concentrations were distinctively lower in birds fed NC vs. PC diets, but increased dramatically with GAA supplementation in Arg-unsupplemented diets, serum citrulline levels may be a way to determine Arg deficiency, and thereby validate the ameliorative effect of GAA by sparing Arg in unsupplemented diets. Homocysteine was similar or slightly lower in muscle than in blood, as corroborated by previous findings (Seo, 2005). The markedly lower levels reported previously (9.66, 4.66, and 7.77 nmol/g homocysteine in liver, breast muscle, and blood samples) might be explained by differences in age of the chickens, as we collected samples from 22-d-old birds as compared to the 31-week-old chicken samples analyzed previously. Additionally, Samuels (2003) reported plasma homocysteine concentrations of 35.9 μM in 5-week-old broilers, which are intermediate concentrations to those quantified in our study and those reported by Seo (2005). As opposed to the various ways dietary GAA supplementation affected concentrations of blood AA and other metabolites, it was interesting to note that plasma homocysteine was neither affected by dietary Arg nor GAA supplementation. Considering GAA receives a labile methyl group from S-adenosylmethionine and the fact that homocysteine is a key biomarker for quantifying methylation demand in the body (Brosnan et al., 2004), we conclude that homocysteine was rapidly metabolized by either remethylation or trans-sulfuration (Brosnan et al., 2004). Overall, it can be concluded from this study that dietary GAA may spare Arg when Arg-deficient diets are fed to young broiler chicks. Our results indicate that 0.12% supplemental GAA was capable of ameliorating the effects of an Arg deficiency on growth performance and muscle phosphagen and glycogen concentrations caused by a dietary Arg deficiency, with outcomes due to this treatment closely matching responses of chicks fed an Arg-adequate diet. 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Google Scholar CrossRef Search ADS PubMed  © 2017 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Efficacy of guanidinoacetic acid on growth and muscle energy metabolism in broiler chicks receiving arginine-deficient diets

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Oxford University Press
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© 2017 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pex378
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Abstract

Abstract Guanidinoacetic acid (GAA) is formed from arginine (Arg) and is the immediate precursor to creatine (Cr) and phosphocreatine (PCr), which are important compounds involved in muscle energy homeostasis. This study sought to determine whether GAA could spare Arg in broiler chicks fed an Arg-deficient practical diet. A basal [0.84% standardized ileal digestible (SID) Arg] was supplemented with combinations of L-Arg (0 or 0.16%) and GAA (0, 0.06, or 0.12%) to form a factorial arrangement of diets; the unsupplemented basal served as the negative control (NC). Additionally, the basal was supplemented with 0.32% Arg to generate an Arg-adequate positive control (PC). Mash diets were fed to 8 replicate pens of 5 chicks per treatment from d 8 to 22 posthatch, with measurements including growth performance, blood GAA metabolites, muscle cellular energy markers, and clinical outcomes. Supplementation of 0.16% Arg increased (P < 0.05) BW gain from d 15 to 22 posthatch, while graded addition of GAA tended to improve BW gain (P < 0.094). Supplementation of either Arg or GAA increased (P < 0.05) feed efficiency from d 15 to 22 and d 8 to 22 posthatch. Birds fed the PC diet had greater (P < 0.05) responses for nearly all blood and tissue outcomes compared with NC-fed birds. Serum GAA was more responsive to supplementation of GAA in the presence versus absence of supplemental Arg (interaction, P < 0.001). Interactions (P < 0.05) were also observed for concentrations of muscle total Cr, creatinine, and most serum essential amino acids, notably Arg. Serum Cr, as well as muscle PCr, total Cr, and glycogen were increased (P < 0.05) independently by Arg and GAA supplementation, with highest levels achieved via combined addition of 0.12% GAA and 0.16% Arg. Minimal effects were detected on hematological and clinical chemistry outcomes. Overall, we conclude that GAA supplementation can spare Arg in broiler chicks fed Arg-deficient practical diets as evidenced by improvements in growth performance and muscle energy stores. INTRODUCTION Volatility in commodity prices and variability in available nutrient content of alternative feedstuffs necessitate careful control over feed formulations for livestock. With increased use of alternative protein sources and a move to lower crude protein concentrations to achieve financial and environmental benefits, the need to incorporate crystalline amino acids (AA) to maintain optimal dietary profiles is rising. Arginine (Arg) is generally considered either the fourth or fifth limiting AA for broiler chickens (Han et al., 1992; Fernandez et al., 1994; Waguespack et al., 2009), but is not currently available in an economically viable form for the animal feed industry. Lower crude protein formulations, increased use of co-product and by-product ingredients such as distillers’ dried grains with solubles (DDGS) (Parsons and Baker, 1983), and a reduction of animal derived protein sources in feed formulations all result in decreased inherent Arg concentrations in the diet of broilers. In contrast, increased growth rate of modern broilers (Havenstein et al., 2003), and a lack of de novo synthesis of Arg (Tamir and Ratner, 1963a), both mandate higher dietary Arg requirements. Therefore, additional dietary Arg sources may be substantial to optimize broiler production in the near future (Han et al., 1992). Arg is an essential AA for broiler growth, but is classified as well as functional AA due to its other roles for broiler nutrition and health (Wu and Morris, 1998). Considering the essentiality of dietary Arg to support lean tissue accretion in the broiler and the lack of a commercially available source of Arg, dietary strategies fueling other metabolic fates of Arg (i.e., for non-protein functions) might be promising solutions to spare Arg, thereby allowing a greater proportion of this AA to be used for muscle protein synthesis. All vertebrate animals generate guanidinoacetic acid (GAA) as an intermediate in the synthesis of creatine (Cr), with the metabolic pathway involving several tissues (Beitz, 2004). In the initial step, GAA is synthesized in the kidney from Arg and glycine (Gly) via Arg: Gly amidinotransferase (AGAT) (Wu and Morris, 1998), and GAA is subsequently imported from the circulation by the liver and converted to Cr. This step is catalyzed by guanidinoacetic acid methyltransferase (GAMT) using S-adenosyl-methionine as a methyl donor, hence producing S-andenosyl homocysteine as a side product (Brosnan et al., 2009; Ostojic et al., 2013). Synthesized Cr is subsequently exported from the liver and transported to target organs, primarily those tissues with high, fluctuating energy demand (i.e., skeletal muscle, heart, brain, retina, spermatozoa), where it is phosphorylated to phosphocreatine (PCr). PCr serves critical physiological functions both as a reserve of high-energy phosphate groups to restore adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and as part of the cellular energy transport system. Due to this central role in cell energy transfer, Cr is present in high concentrations in muscle tissue. However, tissues have a limited storage capacity for Cr and cannot be overloaded (Harris et al., 1992; Greenhaff et al., 1994), so high levels of circulating Cr induce a negative feedback loop on endogenous Cr synthesis by decreasing AGAT expression and thereby decreasing GAA synthesis, most likely in an effort to conserve Arg and methionine (Met) as essential AA for protein synthesis (Wyss and Kaddurah-Daouk, 2000). Creatine synthesis represents a sizeable proportion of whole-body Arg usage, hence providing dietary Cr through use of animal protein ingredients is one strategy to optimize tissue Cr stores, independent of endogenous Cr synthesis, and allow availability of Arg for other functions, namely protein synthesis. However, processed animal protein ingredients available in the feed industry contain low concentrations of Cr (Harris et al., 1997; Dobenecker and Braun, 2015), and crystalline Cr is neither commercially available nor stable through the standard feed manufacturing process (Baker, 2009).Therefore, GAA provided as a crystalline feed additive may be important not only for sparing Arg, but also as the immediate metabolic precursor to Cr, for maintaining overall energy homeostasis in the bird by supporting restoration of cellular ATP concentrations under conditions of high energy turnover (i.e., PCr maintains ATP homeostasis in myofibrils). The Arg-sparing capacity of GAA and Cr has been extensively studied (Edwards et al., 1958; Savage and O’Dell, 1960), but this work was conducted largely using either semi-purified or purified diets. More recent research (Dilger et al., 2013) indicates that GAA improves feed efficiency not only in semi-purified diets deficient in Arg, but also when supplemented in Arg-deficient practical diets. Because the only metabolic fate of GAA is the synthesis of Cr, the Arg-sparing effect of GAA may be due to the decreased need of Arg for GAA and subsequent Cr production. Additionally, there are no safety indications in published literature for dietary GAA fed to broilers. Based on the available evidence that GAA supplementation of Arg-deficient diets elicited an improved growth response, we sought to further investigate the dose-response relationship of GAA on growth performance, blood levels of relevant metabolites (e.g., Cr and Arg), safety outcomes based on clinical pathology and hematological measures, as well as muscle energy stores (ATP, PCr, Cr, and glycogen) when included in practical broiler chicken diets containing varying concentrations of Arg. MATERIALS AND METHODS All animal care procedures were approved by the University of Illinois Institutional Animal Care and Use Committee before initiation of the studies. Animals and Diets Two-hundred eighty male Ross 708 chicks (Hoovers Hatchery, Rudd, IA) were maintained in thermostatically controlled starter batteries with raised-wire floors in an environmentally controlled room with continuous lighting. Water and experimental diets were provided on an ad libitum basis throughout the study. Chicks arrived at 2 d posthatch and received a diet adequate in all nutrients (National Research Council, 1994) from d 2 to 7 posthatch. Following an overnight fast, chicks were weighed, wing-banded, and assigned to dietary treatments on d 8 posthatch in a randomized complete block design such that the average initial pen weights were not different among treatments. Eight replicate pens of 5 chicks received one of 7 treatment diets during a 14-d feeding study (d 8 to 22 posthatch). Battery pens (99.1 cm long, 33.7 cm wide, 26.7 cm high) provided 666.8 cm2 of floor space and access to 13.5 and 6.7 lineal cm/bird of feeder and water space, respectively, via hanging troughs in a room with adequate lighting, ventilation, and temperature control to meet agricultural standards. Chicks and feeders were weighed on d 8, 15, and 22 posthatch, and BW gain, feed intake, and G:F were calculated for each replicate pen of chicks. An Arg-deficient basal, formulated to contain 0.84% standardized ileal digestible (SID) Arg, was manufactured, composed primarily of corn, soybean meal, DDGS, and corn gluten meal (Table 1). Vitamin and mineral premixes, as well as crystalline AA, were incorporated into the basal to meet or exceed requirements for broiler chicks (National Research Council, 1994), with the exception of Arg (Table 2). Experimental treatment diets were subsequently produced by supplementing the basal with 2 levels of Arg (0 or 0.16% to provide 0.84 or 1.00% formulated SID Arg) and 3 levels of GAA (0, 0.06, or 0.12%) (CreAMINO®, GAA, 96% min, AlzChem AG, Trostberg, Germany) at the expense of corn to form a 2 × 3 factorial arrangement of dietary treatments (6 treatments). The unsupplemented basal diet (i.e., containing 0% supplementation of both Arg and GAA) served as the negative control (NC) based on established recommendations for dietary Arg (NRC, 1994). A seventh dietary treatment was produced by supplementing the basal diet with 0.32% Arg (1.16% SID Arg) to serve as the positive control (PC). All diets were fed in mash form in a single feeding phase that lasted from d 8 to 22 posthatch. Table 1. Basal diet formulation and nutrient composition. Ingredient, %    Value    Corn    57.57    Soybean Meal    10.03    DDGS    12.04    Corn Gluten Meal    11.54    Soy Oil    3.01    Salt    0.40    Limestone    1.50    Dicalcium Phosphate    1.71    Vitamin Premix1    0.20    Mineral Premix2    0.15    Choline Chloride    0.20    Titanium Dioxide    0.40    L-Lysine HCl    0.70    DL-Methionine    0.25    L-Isoleucine    0.06    L-Threonine    0.18    L-Tryptophan    0.05    Calculated Proximates       Crude Protein, %    21.3     Calcium, %    10.0     Phosphorus (total), %    6.8     Phosphorus (available), %    4.5     AMEN, kcal/kg    3148    Calculated Amino Acids, %  Total    SID3   Arg  0.96    0.84   Ile  0.86    0.76   Leu  2.53    2.31   Lys  1.24    1.14   Met  0.68    0.64   Met+Cys  1.04    0.92   Thr  0.87    0.73   Trp  0.23    0.19   Val  1.00    0.86  Ingredient, %    Value    Corn    57.57    Soybean Meal    10.03    DDGS    12.04    Corn Gluten Meal    11.54    Soy Oil    3.01    Salt    0.40    Limestone    1.50    Dicalcium Phosphate    1.71    Vitamin Premix1    0.20    Mineral Premix2    0.15    Choline Chloride    0.20    Titanium Dioxide    0.40    L-Lysine HCl    0.70    DL-Methionine    0.25    L-Isoleucine    0.06    L-Threonine    0.18    L-Tryptophan    0.05    Calculated Proximates       Crude Protein, %    21.3     Calcium, %    10.0     Phosphorus (total), %    6.8     Phosphorus (available), %    4.5     AMEN, kcal/kg    3148    Calculated Amino Acids, %  Total    SID3   Arg  0.96    0.84   Ile  0.86    0.76   Leu  2.53    2.31   Lys  1.24    1.14   Met  0.68    0.64   Met+Cys  1.04    0.92   Thr  0.87    0.73   Trp  0.23    0.19   Val  1.00    0.86  1Provided per kg of diet: retinyl acetate, 4,400 IU; cholecalciferol, 25 μg; DL-a- tocopheryl acetate, 11 IU; vitamin B12, 0.01 mg; riboflavin, 4.41 mg; D-Ca-pantothenate, 10 mg; niacin, 22 mg; menadione sodium bisulfite complex, 2.33 mg. 2Provided as milligrams per kg of diet: Mn, 75 from MnO; Fe, 75 from FeSO4 • 7H2O; Zn, 75 from ZnO; Cu, 5 from CuSO4 • 5H2O; I, 0.75 from ethylene diamine dihydroiodide; Se, 0.1 from Na2SeO3. 3Standardized ileal digestible AA composition calculated using data acquired from AMINODat® 4.0 (Evonik Industries AG, Hanau-Wolfgang, Germany). View Large Table 2. Analyzed composition of dietary treatments (as-is basis).   Dietary Treatment1  Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32  Nutrient GAA, %  0.002  0.06  0.12  0.00  0.06  0.12  0.003  Dry Matter, %  88.8  89.0  89.0  88.9  89.2  89.1  89.0  Crude Protein, %  21.4  21.9  20.7  21.8  22.2  21.4  23.0  Crude Fat, %  5.8  6.2  6.0  6.1  6.0  6.3  5.9  Crude Fiber, %  2.3  2.1  2.3  2.2  2.1  2.2  2.2  Ash, %  6.0  6.1  5.8  6.1  6.4  5.8  6.5  GAA, mg/kg4  <1.0  593  1160  <1.0  563  1130  <1.0  Creatine, mg/kg4  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  Folic Acid, mg/kg  0.94  0.87  0.97  0.85  1.00  1.08  0.92  Choline, mg/kg  1030  1160  1210  1110  1190  1080  1070  Betaine, mg/kg  6800  4900  7500  7300  6500  4000  7200  Total Amino Acids, %5  Essential   Arg  1.01  1.01  1.01  1.16  1.16  1.15  1.31   His  0.52  0.52  0.51  0.52  0.52  0.50  0.52   Ile  0.86  0.85  0.85  0.86  0.85  0.83  0.86   Leu  2.49  2.50  2.45  2.50  2.46  2.41  2.49   Lys  1.21  1.22  1.25  1.15  1.22  1.29  1.22   Met  0.68  0.64  0.66  0.66  0.65  0.68  0.66   Phe  1.10  1.11  1.09  1.11  1.09  1.07  1.10   Thr  0.87  0.91  0.85  0.90  0.90  0.88  0.89   Val  0.96  0.95  0.94  0.95  0.96  0.93  0.96  Non-Essential   Ala  1.45  1.45  1.41  1.45  1.42  1.39  1.43   Asp  1.55  1.56  1.56  1.57  1.57  1.53  1.56   Cys  0.38  0.38  0.37  0.38  0.38  0.37  0.38   Glu  3.87  3.89  3.83  3.90  3.85  3.77  3.87   Gly  0.76  0.76  0.76  0.76  0.76  0.75  0.76   Pro  1.57  1.55  1.53  1.57  1.55  1.51  1.56   Ser  1.02  1.03  1.00  1.03  1.01  1.00  1.01    Dietary Treatment1  Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32  Nutrient GAA, %  0.002  0.06  0.12  0.00  0.06  0.12  0.003  Dry Matter, %  88.8  89.0  89.0  88.9  89.2  89.1  89.0  Crude Protein, %  21.4  21.9  20.7  21.8  22.2  21.4  23.0  Crude Fat, %  5.8  6.2  6.0  6.1  6.0  6.3  5.9  Crude Fiber, %  2.3  2.1  2.3  2.2  2.1  2.2  2.2  Ash, %  6.0  6.1  5.8  6.1  6.4  5.8  6.5  GAA, mg/kg4  <1.0  593  1160  <1.0  563  1130  <1.0  Creatine, mg/kg4  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  <1.0  Folic Acid, mg/kg  0.94  0.87  0.97  0.85  1.00  1.08  0.92  Choline, mg/kg  1030  1160  1210  1110  1190  1080  1070  Betaine, mg/kg  6800  4900  7500  7300  6500  4000  7200  Total Amino Acids, %5  Essential   Arg  1.01  1.01  1.01  1.16  1.16  1.15  1.31   His  0.52  0.52  0.51  0.52  0.52  0.50  0.52   Ile  0.86  0.85  0.85  0.86  0.85  0.83  0.86   Leu  2.49  2.50  2.45  2.50  2.46  2.41  2.49   Lys  1.21  1.22  1.25  1.15  1.22  1.29  1.22   Met  0.68  0.64  0.66  0.66  0.65  0.68  0.66   Phe  1.10  1.11  1.09  1.11  1.09  1.07  1.10   Thr  0.87  0.91  0.85  0.90  0.90  0.88  0.89   Val  0.96  0.95  0.94  0.95  0.96  0.93  0.96  Non-Essential   Ala  1.45  1.45  1.41  1.45  1.42  1.39  1.43   Asp  1.55  1.56  1.56  1.57  1.57  1.53  1.56   Cys  0.38  0.38  0.37  0.38  0.38  0.37  0.38   Glu  3.87  3.89  3.83  3.90  3.85  3.77  3.87   Gly  0.76  0.76  0.76  0.76  0.76  0.75  0.76   Pro  1.57  1.55  1.53  1.57  1.55  1.51  1.56   Ser  1.02  1.03  1.00  1.03  1.01  1.00  1.01  1The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. Abbreviations: GAA, guanidinoacetic acid. 2Negative control treatment. 3Positive control treatment. 4Analytical limit of detection was 1.0 mg/kg. 5Amino acids values were standardized to a dry matter content of 88%. View Large Sample Collection On d 22 posthatch, one non-fasted bird per pen was randomly chosen and euthanized via an intracardiac injection of 390 mg/mL sodium pentobarbital at 0.2 mL/kg BW to facilitate rapid collection of breast muscle tissue. A muscle biopsy sample, ranging from 1 to 5 g of wet tissue, was collected within 30 s of euthanasia and immediately immersed directly in liquid nitrogen until gas elaboration ceased. Time from euthanasia to flash-freezing of the muscle biopsy was no more than 60 s for any individual bird in order to prevent enzymatic PCr degradation. Snap-frozen muscle biopsy samples were then shattered using blunt force, with frozen muscle aliquots randomly dispensed into pre-cooled cryovials and placed back in liquid nitrogen until transferred to storage at −80°C. Once rapid muscle sample collection was complete, the bird was then passed to another station for additional collection of breast muscle, where the complete left pectoralis major muscle was removed by hand and placed into a pre-labeled storage bag. These samples were ultimately stored at −20°C until analysis of GAA and homocysteine as described below. Upon successful muscle collection, all remaining birds in each pen were euthanized by CO2 asphyxiation. Immediately after euthanasia, blood was separately collected from 2 of the remaining non-fasted birds per pen (randomly selected) via intracardiac puncture into evacuated tubes containing no anticoagulant, ethylenediaminetetraacetic acid (EDTA), or heparin to preserve serum, plasma, and whole-blood samples, respectively. Blood samples for plasma were stored on ice for no more than 1 h, and serum was allowed to clot at room temperature for no more than 2 h, before sample processing procedures began. Blood tubes for plasma and serum were centrifuged at 1,300 × g while being held at 4°C or 20°C, respectively, and all plasma and serum aliquots were subsequently stored at −80°C. Whole-blood samples were kept on ice until being stored at 4°C pending analysis. Following blood collection and processing steps, serum, plasma, and whole-blood samples from the 2 birds per pen were pooled into composite samples. Sample Analyses Diets were analyzed for dry matter, crude fat, crude fiber, and ash using standardized methods (AOAC International, 2006). Total nitrogen was determined using a Leco analyzer (TruMac N, Leco Corp., St. Joseph, MO) standardized with EDTA (method 990.03, AOAC International, 2006). Dietary AA concentrations were determined by ion-exchange chromatography with postcolumn derivatization with ninhydrin. Amino acids were oxidized with performic acid, which was neutralized with Na metabisulfite (Llames and Fontaine, 1994; Commission Directive, 1998). Amino acids were liberated from the protein by hydrolysis with 6 N HCl for 24 h at 110°C and quantified with the internal standard by measuring the absorption of reaction products with ninhydrin at 570 nm. Tryptophan (Trp) was determined by high-performance liquid chromatography (HPLC) with fluorescence detection (extinction 280 nm, emission 356 nm), after alkaline hydrolysis with barium hydroxide octahydrate for 20 h at 110°C (Commission Directive, 2000). Tyrosine (Tyr) was not determined. Dietary folic acid, choline, and betaine were analyzed using validated methods (AOAC International, 2006) by an analytical laboratory (Eurofins Scientific Inc., Des Moines, IA). Dietary concentrations of GAA and Cr were quantified using fully validated procedures (Dobenecker and Braun, 2015) by an analytical laboratory (AlzChem AG, Trostberg, Germany). Muscle PCr, free Cr, and ATP concentrations were quantified using fully validated procedures (Harris et al., 1974; Swiss BioQuant, Reinach, Switzerland). In brief, muscle biopsy samples were freeze-dried, powdered, and following extraction with 1 mM EDTA in 0.5 M ice-cold perchloric acid (PCA) an aliquot of 25 μL of neutralized PCA extract (corresponding to 25 mg of dried muscle) was used for the simultaneous determination of ATP and PCr. For determination of free Cr, the neutralized extract was diluted with water (1:2 to 1:5 dilution). The analytical method was based on enzymatic determinations, which ultimately resulted in either reduction of NADP to NADPH (for ATP and PCr) or oxidation of NADH to NAD (for free Cr). For determination of ATP, 25 μL of the neutralized extract were mixed with 225 μL of TEA Buffer 1 (10 mM magnesium acetate, 1 mM EDTA, 1 mM DTT, 1 mM NADP, 0.04 mM ADP, 5 mM glucose, 4 μg/mL glucose-6-phosphate dehydrogenase in 100 mM TEA × HCl, pH 7.5) and the increase of absorbance at 340 nm was measured. Subsequently 10 μL creatine phosphokinase (5 mg/mL) was added to determine PCr based on the absorbance increase at 340 nm. For determination of Cr, 25 μL of the neutralized diluted extract were mixed with 225 μL of TEA Buffer 2 (10 mM magnesium acetate, 1 mM EDTA, 30 mM KCl, 1 mM phosphoenol pyruvate, 0.3 mM NADH, 2 mM ATP, 40 μg/mL pyruvate kinase, 20 μg/mL lactate dehydrogenase in 100 mM TEA × HCl, pH 8.5). After addition of 10 μL creatine phosphokinase (15 mg/mL in 0.5% NaHCO3, 0.05% BSA) the decrease of absorbance at 340 nm was measured. ATP, PCr and Cr contents were calculated based on the molar absorption coefficient for NADH at 340 nm (6.22 mM–1 × cm–1). Muscle glycogen concentrations were quantified using fully validated procedures (Swiss BioQuant, Reinach, Switzerland). In brief, 10 mg of freeze-dried muscle was hydrolyzed in 0.5 mL of 1 M HCl for 2 h at approximately 99°C to convert glycogen to glucose. The hydrolysate was then neutralized by the addition of 0.15 mL (per 0.5 mL hydrolysate) of 0.1 M imidazol maintained at pH 7.0 using 0.1 M NaOH. Following centrifugation at 13,000 × g for 5 min, the supernatant was diluted 1:20 with water. Subsequently, the supernatants were subjected to a glucose analysis based on the total hydrolysis of glycogen to glucose by enzymatic determination using a commercially available glucose oxidase assay (GAGO-20; Sigma-Aldrich, St. Louis, MO). Homocysteine was determined in wet muscle tissue by ion chromatography (Dionex DX500, with gradient pump and fluorescence detector) using a fully validated procedure (LiChrospher 100 RP-18 column, 4.6 × 250 mm, 5 μm; column temperature 30°C, flow rate set at 1.0 mL min–1). In brief, 100 mg of the minced wet tissue sample was weighed into a reaction flask, 400 μL phosphate salt buffer (pH 7.4) was added, and the suspension homogenized thoroughly using a rotor-stator mixer. An aliquot of the homogenate was reacted with tri-n-butylphosphine solution at 4°C, and after addition of 0.6 M perchloric acid, the suspension was centrifuged. The supernatant was subsequently mixed with borate buffer (pH 10.5) and SBDF reagent (ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, 1 g/L in borate buffer), and the resulting solution was heated for 60 min at 60°C and then centrifuged for 10 min at 20,000 rpm after cooling to room temperature. Finally, the supernatant was used for quantification of homocysteine, using a mobile phased produced by dissolving 5.86 g sodium acetate in water in a 1-L volumetric flask, and subsequently adding 1.72 g glacial acetic acid and 20 mL methanol prior to filling the volumetric flask to volume with water. Serum [AA, Cr, creatinine (Crn), GAA] and plasma samples (homocysteine) were analyzed using standardized procedures (Baylor University, Houston, TX). Whole blood was submitted to the University of Illinois Urbana-Champaign Veterinary Diagnostic Laboratory for analysis of hematological and clinical pathology parameters. Blood biochemistry was assayed using an automated spectrophotometric method on a Hitachi 917 analyzer (Roche, Indianapolis, IN), while hematological parameters were assessed using a combination of automated and manual procedures. Statistical Analysis Data from all diets except the PC were analyzed as a 2-way analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Inst., Cary, NC); dietary supplemental Arg and GAA concentrations were independent variables in the statistical model. Whereas birds were allotted based on a randomized complete block design, the fixed effect of block was insignificant in all cases and was therefore removed from the statistical model. When interactive effects were noted, means separation was conducted using a Tukey's adjustment. In addition to the 2-way ANOVA, each dietary treatment was compared to the PC diet using a 2-tailed Dunnett's test. Overall treatment effects with a probability of P < 0.05 were accepted as statistically significant. RESULTS Overall, formulation objectives were achieved in terms of creating an Arg-deficient basal diet, and graded supplementation of Arg and GAA was realized in the final diets (Table 2). Mortality was only 1.43% and was not affected by dietary treatment. Growth Performance Feed intake was not affected by any of the dietary treatments and no interactive effects were noted for any performance parameter in this study. Body weight gain d 15 to 22 and d 8 to 22 posthatch were increased (P < 0.05) due to the main effect of Arg supplementation, with the NC diet (0% supplemental Arg and GAA) exhibiting reduced (P < 0.05) BW gain compared with the PC diet for d 15 to 22 (Table 3). No main effects of GAA supplementation was observed for BW gain regardless of feeding period, though a trend for GAA to increase BW gain at d 15 to 22 was observed (P = 0.094). Feed efficiency (i.e., G:F) was independently increased (P < 0.05) due to supplementation of either 0.16% Arg or graded dietary GAA (i.e., main effects of both Arg and GAA) from d 15 to 22 and d 8 to 22 posthatch to reach similar performance as elicited by the PC diet. Table 3. Growth performance of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Body weight, g   d8  99.3  99.4  99.4  99.3  99.5  99.5  99.3  2.83  1.00  1.00  1.00   d15  299.8  285.4  286.3  293.6  299.7  300.4  301.1  10.02  0.63  0.90  0.51   d22  627.9  626.2  641.0  642.0  656.3  662.2  668.5  16.37  0.076  0.59  0.89  BW gain, g/chick   d 8 to 15  200.5  186.0  186.9  194.3  200.2  200.9  201.8  7.52  0.44  0.83  0.31   d 15 to 22  328.1*  340.9  354.7  348.4  356.6  361.8  367.4  8.99  0.008  0.094  0.76   d 8 to 22  528.6  526.8  541.6  542.7  556.8  562.7  569.2  14.39  0.037  0.52  0.86  Feed intake, g/chick   d 8 to 15  301.4  278.9  278.6  282.1  295.1  283.1  286.9  9.37  0.85  0.47  0.13   d 15 to 22  514.0  516.3  512.4  509.4  518.7  503.3  523.6  13.60  0.77  0.75  0.90   d 8 to 22  815.6  797.7  791.0  791.5  813.8  791.3  810.8  20.98  0.98  0.73  0.59  G:F, g/kg   d 8 to 15  663.8  666.8  668.9  687.0  680.9  711.4  697.4  17.94  0.100  0.57  0.69   d 15 to 22  637.9*  661.4  693.0  683.9  690.5  718.1  701.9  15.43  0.003  0.011  0.75   d 8 to 22  647.3*  661.5  684.3  685.1  686.9  710.9  700.5  13.63  0.004  0.047  0.87    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Body weight, g   d8  99.3  99.4  99.4  99.3  99.5  99.5  99.3  2.83  1.00  1.00  1.00   d15  299.8  285.4  286.3  293.6  299.7  300.4  301.1  10.02  0.63  0.90  0.51   d22  627.9  626.2  641.0  642.0  656.3  662.2  668.5  16.37  0.076  0.59  0.89  BW gain, g/chick   d 8 to 15  200.5  186.0  186.9  194.3  200.2  200.9  201.8  7.52  0.44  0.83  0.31   d 15 to 22  328.1*  340.9  354.7  348.4  356.6  361.8  367.4  8.99  0.008  0.094  0.76   d 8 to 22  528.6  526.8  541.6  542.7  556.8  562.7  569.2  14.39  0.037  0.52  0.86  Feed intake, g/chick   d 8 to 15  301.4  278.9  278.6  282.1  295.1  283.1  286.9  9.37  0.85  0.47  0.13   d 15 to 22  514.0  516.3  512.4  509.4  518.7  503.3  523.6  13.60  0.77  0.75  0.90   d 8 to 22  815.6  797.7  791.0  791.5  813.8  791.3  810.8  20.98  0.98  0.73  0.59  G:F, g/kg   d 8 to 15  663.8  666.8  668.9  687.0  680.9  711.4  697.4  17.94  0.100  0.57  0.69   d 15 to 22  637.9*  661.4  693.0  683.9  690.5  718.1  701.9  15.43  0.003  0.011  0.75   d 8 to 22  647.3*  661.5  684.3  685.1  686.9  710.9  700.5  13.63  0.004  0.047  0.87  *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. View Large Breast Muscle Analysis Tissue metabolite concentrations were expressed both as absolute concentrations as well as relative concentrations (Table 4), normalized to ATP in order to standardize for lean muscle tissue as suggested by Harris et al. (1992). Birds fed the NC diet exhibited lower (P < 0.05) absolute and relative concentrations of muscle PCr, as well as absolute total Cr, when compared to birds fed the PC diet. Muscle absolute and relative PCr concentrations independently increased (P < 0.05) due to dietary addition of either 0.16% Arg or graded GAA supplementation, but no interactive effects were noted. Highest metabolite concentrations were obtained from supplementation of the basal diet with both 0.16% Arg and 0.12% GAA, which resulted in absolute concentrations of muscle PCr being increased (P < 0.05) by 189% and 46% compared with the NC and PC diets, respectively. Similarly, the PCr: ATP ratio was increased by 227% and 72% compared with the NC and PC diets. Graded GAA supplementation increased (P < 0.05) muscle total Cr concentrations, but the effect was more pronounced in diets containing supplemental Arg (interaction, P < 0.05). The addition of 0.16% Arg and 0.12% GAA resulted in total Cr concentrations that were increased (P < 0.05) by 26% compared with the PC diet. Muscle glycogen was independently increased (main effects; P < 0.05) by supplementation of either Arg or GAA. Muscle GAA was measured but not reported due to most samples containing GAA concentrations below the detection limit (<5 mg/kg). Homocysteine concentrations in wet muscle were not affected by dietary treatment. Table 4. Muscle analyses of energy-related metabolites of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Absolute concentrations   ATP, mmol/kg DW  37.8  38.0  41.0  38.1  40.8  34.1  40.1  2.36  0.62  0.71  0.11   PCr, mmol/kg DW  37.6*  56.1  91.0  61.7  82.4  108.7*  74.4  8.14  0.001  <0.001  0.86   Free Cr, mmol/kg DW  68.9  56.3  59.5  54.7  76.5  67.3  64.7  7.81  0.74  0.84  0.094   Total Cr, mmol/kg DW7  106.6a*  112.4a*  150.6b  116.5a  158.9b  176.0b*  139.2  7.13  <0.001  <0.001  0.044   Glycogen, mmol/kg DW  171.5  221.9  260.8  239.8  266.1  310.1  265.0  26.75  0.008  0.010  0.88  Relative concentrations   PCr:ATP ratio  0.99*  1.54  2.20  1.68  2.04  3.24*  1.88  0.24  <0.001  <0.001  0.52   Free Cr:ATP ratio  1.92  1.58  1.52  1.45  1.95  2.03  1.66  0.27  0.82  0.94  0.15   Total Cr:ATP ratio  2.91  3.11  3.73  3.12  4.00  5.27*  3.55  0.30  0.001  <0.001  0.091   PCr/total Cr, %  34.5  51.0  60.5  52.2  51.9  61.7  53.7  5.40  0.11  0.007  0.22    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Absolute concentrations   ATP, mmol/kg DW  37.8  38.0  41.0  38.1  40.8  34.1  40.1  2.36  0.62  0.71  0.11   PCr, mmol/kg DW  37.6*  56.1  91.0  61.7  82.4  108.7*  74.4  8.14  0.001  <0.001  0.86   Free Cr, mmol/kg DW  68.9  56.3  59.5  54.7  76.5  67.3  64.7  7.81  0.74  0.84  0.094   Total Cr, mmol/kg DW7  106.6a*  112.4a*  150.6b  116.5a  158.9b  176.0b*  139.2  7.13  <0.001  <0.001  0.044   Glycogen, mmol/kg DW  171.5  221.9  260.8  239.8  266.1  310.1  265.0  26.75  0.008  0.010  0.88  Relative concentrations   PCr:ATP ratio  0.99*  1.54  2.20  1.68  2.04  3.24*  1.88  0.24  <0.001  <0.001  0.52   Free Cr:ATP ratio  1.92  1.58  1.52  1.45  1.95  2.03  1.66  0.27  0.82  0.94  0.15   Total Cr:ATP ratio  2.91  3.11  3.73  3.12  4.00  5.27*  3.55  0.30  0.001  <0.001  0.091   PCr/total Cr, %  34.5  51.0  60.5  52.2  51.9  61.7  53.7  5.40  0.11  0.007  0.22  a-bMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: ATP = adenosine triphosphate, Cr = creatine, DW = dry weight, GAA = guanidinoacetic acid, PCr = phosphocreatine, WW = wet weight. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. 7Calculated as PCr plus free Cr. View Large Blood Analysis Serum Arg concentrations were unchanged by graded GAA in diets containing 0% added Arg, but increased 38% due to addition of 0.12% GAA in diets containing 0.16% added Arg (interaction, P < 0.05; Table 5). Interactive effects (P < 0.05) were also observed for histidine (His), isoleucine (Ile), lysine (Lys), phenylalanine (Phe), and valine (Val), which decreased an average of 34 and 7% due to addition of 0.12% GAA when included in diets containing 0.0 or 0.16% added Arg, respectively. Glutamine (Gln) was the only non-essential AA that exhibited an interaction (P < 0.05), with all other non-essential AA (except asparagine [Asn]) decreasing (P < 0.05) due to Arg supplementation. Additionally, alanine (Ala) decreased (P < 0.05) due to graded addition of GAA, regardless of dietary Arg concentration. Table 5. Serum amino acid and metabolite concentrations (μM) of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Essential   Arginine  155.8a,b,*  136.9a*  143.8a*  199.1b,c,*  244.6c,d,*  274.0d*  364.9  15.35  <0.001  0.13  0.019   Histidine  127.0c*  109.1b,c  79.6a,b  80.9a,b  74.1a,b  73.4a  84.7  8.03  <0.001  0.005  0.046   Isoleucine  118.4b*  89.4a  82.9a  79.6a  82.9a  76.8a  77.3  4.26  <0.001  0.001  0.001   Leucine  434.3*  354.4  334.5  370.3  337.4  318.4  341.1  16.92  0.002  0.001  0.28   Lysine  579.0c*  470.6b,c*  272.5a  297.3a,b  237.8a  262.5a  274.3  43.58  <0.001  0.001  0.007   Methionine  196.4  181.3  179.6  177.0  158.8  162.0  161.6  10.64  0.017  0.22  0.97   Phenylalanine  217.5  184.1  184.4  188.3  198.8  186.1  172.9  8.67  0.021  0.13  0.042   Threonine  2093*  1839*  1333  1410  1243  1175  1254  113.18  <0.001  0.001  0.054   Valine  245.1b*  173.3a  160.9a  164.3a  158.3a  148.6a  150.8  8.33  <0.001  <0.001  0.001  Non-Essential   Alanine  1328*  1148  1077  1055  991.1  950.1  927.3  71.08  0.001  0.047  0.56   Asparagine  20.9  43.5  60.3  41.8  59.6  35.0  44.6  21.79  0.86  0.62  0.51   Aspartic acid  221.9*  220.6*  185.6  176.1  162.6  179.1  157.1  13.32  0.001  0.46  0.14   Cysteine  85.6*  80.9  75.1  75.0  75.8  72.5  72.9  3.34  0.021  0.15  0.48   Glutamine  1,243b  1,514c*  1,180b  1,095a,b  1,070a,b  882.6a*  1095  54.66  <0.001  <0.001  0.033   Glutamic acid  266.0*  250.5  254.4  224.9  229.9  254.4  224.5  9.43  0.009  0.32  0.10   Glycine  731.3*  696.3*  632.0*  520.5  521.8  517.0  476  38.27  <0.001  0.40  0.46   Proline  832.8  883.9  886.4  756.3  748.0  745.3  768.5  52.37  0.027  0.90  0.79   Serine  936.5*  918.0*  800.0  721.9  680.5  652.3  715.0  48.08  <0.001  0.098  0.63   Tyrosine  409.6  422.0  365.0  329.9  363.6  337.5  326.9  26.80  0.024  0.31  0.62  Other Metabolites   Alpha-aminobutyric acid  71.9c,*  34.8a,*  50.5b,*  42.5a,b,*  37.5a,b,*  36.4a,b,*  20.4  3.31  <0.001  <0.001  <0.001   Citrulline  2.75a,*  9.0b  15.9c,*  15.8c,*  14.9c,*  16.8c,*  9.5  1.07  <0.001  <0.001  <0.001   Ornithine  11.0*  7.0*  7.38*  20.1*  17.3*  17.6*  28.0  1.78  <0.001  0.12  0.94   1-Methylhistidine  38.1*  30.4  24.6  26.6  27.4  22.8  20.5  2.87  0.001  0.014  0.20   3-Methylhistidine  16.3c,*  12.6b  7.13a  9.25a,b  7.25a  8.88a,b  11.0  1.05  0.001  0.001  0.001   Phosphoserine  20.6a  19.0a  70.8b,*  75.9b,*  75.5b,*  68.9b,*  20.6  3.47  <0.001  <0.001  <0.001   Phosphoethanolamine  2.75a,b  2.25a  4.13a,b,*  14.9c,*  4.38b,*  4.50b,*  1.63  0.48  <0.001  <0.001  <0.001   Taurine  362.9  297.8  340.5  381  288.8  311.8  286.9  29.82  0.074  0.038  0.73    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Essential   Arginine  155.8a,b,*  136.9a*  143.8a*  199.1b,c,*  244.6c,d,*  274.0d*  364.9  15.35  <0.001  0.13  0.019   Histidine  127.0c*  109.1b,c  79.6a,b  80.9a,b  74.1a,b  73.4a  84.7  8.03  <0.001  0.005  0.046   Isoleucine  118.4b*  89.4a  82.9a  79.6a  82.9a  76.8a  77.3  4.26  <0.001  0.001  0.001   Leucine  434.3*  354.4  334.5  370.3  337.4  318.4  341.1  16.92  0.002  0.001  0.28   Lysine  579.0c*  470.6b,c*  272.5a  297.3a,b  237.8a  262.5a  274.3  43.58  <0.001  0.001  0.007   Methionine  196.4  181.3  179.6  177.0  158.8  162.0  161.6  10.64  0.017  0.22  0.97   Phenylalanine  217.5  184.1  184.4  188.3  198.8  186.1  172.9  8.67  0.021  0.13  0.042   Threonine  2093*  1839*  1333  1410  1243  1175  1254  113.18  <0.001  0.001  0.054   Valine  245.1b*  173.3a  160.9a  164.3a  158.3a  148.6a  150.8  8.33  <0.001  <0.001  0.001  Non-Essential   Alanine  1328*  1148  1077  1055  991.1  950.1  927.3  71.08  0.001  0.047  0.56   Asparagine  20.9  43.5  60.3  41.8  59.6  35.0  44.6  21.79  0.86  0.62  0.51   Aspartic acid  221.9*  220.6*  185.6  176.1  162.6  179.1  157.1  13.32  0.001  0.46  0.14   Cysteine  85.6*  80.9  75.1  75.0  75.8  72.5  72.9  3.34  0.021  0.15  0.48   Glutamine  1,243b  1,514c*  1,180b  1,095a,b  1,070a,b  882.6a*  1095  54.66  <0.001  <0.001  0.033   Glutamic acid  266.0*  250.5  254.4  224.9  229.9  254.4  224.5  9.43  0.009  0.32  0.10   Glycine  731.3*  696.3*  632.0*  520.5  521.8  517.0  476  38.27  <0.001  0.40  0.46   Proline  832.8  883.9  886.4  756.3  748.0  745.3  768.5  52.37  0.027  0.90  0.79   Serine  936.5*  918.0*  800.0  721.9  680.5  652.3  715.0  48.08  <0.001  0.098  0.63   Tyrosine  409.6  422.0  365.0  329.9  363.6  337.5  326.9  26.80  0.024  0.31  0.62  Other Metabolites   Alpha-aminobutyric acid  71.9c,*  34.8a,*  50.5b,*  42.5a,b,*  37.5a,b,*  36.4a,b,*  20.4  3.31  <0.001  <0.001  <0.001   Citrulline  2.75a,*  9.0b  15.9c,*  15.8c,*  14.9c,*  16.8c,*  9.5  1.07  <0.001  <0.001  <0.001   Ornithine  11.0*  7.0*  7.38*  20.1*  17.3*  17.6*  28.0  1.78  <0.001  0.12  0.94   1-Methylhistidine  38.1*  30.4  24.6  26.6  27.4  22.8  20.5  2.87  0.001  0.014  0.20   3-Methylhistidine  16.3c,*  12.6b  7.13a  9.25a,b  7.25a  8.88a,b  11.0  1.05  0.001  0.001  0.001   Phosphoserine  20.6a  19.0a  70.8b,*  75.9b,*  75.5b,*  68.9b,*  20.6  3.47  <0.001  <0.001  <0.001   Phosphoethanolamine  2.75a,b  2.25a  4.13a,b,*  14.9c,*  4.38b,*  4.50b,*  1.63  0.48  <0.001  <0.001  <0.001   Taurine  362.9  297.8  340.5  381  288.8  311.8  286.9  29.82  0.074  0.038  0.73  a-dMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. View Large Serum concentrations of citrulline, phosphoserine, and phosphoethanolamine increased due to GAA addition to Arg-unsupplemented diets, but were unchanged when GAA was added to diets containing supplemental Arg (interaction, P < 0.05). Opposite interactive effects (P < 0.05) were observed for alpha-aminobutyric acid and 3-methylhistidine, which decreased due to GAA addition to Arg-unsupplemented diets, but were unchanged when GAA was added to diets containing supplemental Arg. Serum ornithine increased (P < 0.05) 117% due to Arg supplementation (i.e., PC vs. NC), and a main effect (P < 0.05) of GAA supplementation caused a decrease in taurine concentrations. Finally, serum 1-methylhistidine decreased (P < 0.05) by 18 and 27% due to supplementation with either 0.16% Arg or 0.12% GAA, respectively. In general, the NC diet elicited lower serum Cr concentrations as compared with birds fed the PC diet, while serum GAA and plasma homocysteine were not different between NC- and PC-fed birds. Graded GAA supplementation increased (P < 0.05) serum GAA concentrations, but the effect was more pronounced in diets containing 0.16% supplemental Arg (interaction, P < 0.05; Table 6). Moreover, addition of either 0.06 or 0.12% GAA increased (P < 0.05) serum GAA by at least 323% when compared with the PC diet. Serum Cr concentrations independently increased (P < 0.05) due to dietary addition of either Arg or GAA, and no interactive effect was observed. Serum Crn was analyzed to be below detection limits (<4.4 μM) in all samples, and therefore data are not available, and plasma homocysteine was not affected by dietary treatment. Table 6. Blood and muscle concentrations of creatine-related metabolites from chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Plasma   Homocysteine, μM  155.4  151.6  150.5  178.2  170.5  157.7  134.4  13.42  0.057  0.64  0.83   Uric acid, μM  8.5a*  8.3a*  6.5a,b  6.2a,b  5.7b  6.9a,b  5.9  0.59  0.002  0.52  0.028  Serum   Creatine, μM  14.2*  22.3  37.1  19.8  26.0  43.2*  28.3  3.31  0.006  <0.001  0.93   Guanidinoacetic acid, μM  0.39d  3.85c*  9.84b*  0.48d  4.91c*  13.95a*  0.91  0.54  <0.001  <0.001  0.001  Muscle7   Homocysteine, μmol/kg8  75.3  114.3  82.7  108.6  78.3  70.9  119.0  39.96  0.28  0.36  0.054    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Plasma   Homocysteine, μM  155.4  151.6  150.5  178.2  170.5  157.7  134.4  13.42  0.057  0.64  0.83   Uric acid, μM  8.5a*  8.3a*  6.5a,b  6.2a,b  5.7b  6.9a,b  5.9  0.59  0.002  0.52  0.028  Serum   Creatine, μM  14.2*  22.3  37.1  19.8  26.0  43.2*  28.3  3.31  0.006  <0.001  0.93   Guanidinoacetic acid, μM  0.39d  3.85c*  9.84b*  0.48d  4.91c*  13.95a*  0.91  0.54  <0.001  <0.001  0.001  Muscle7   Homocysteine, μmol/kg8  75.3  114.3  82.7  108.6  78.3  70.9  119.0  39.96  0.28  0.36  0.054  a-dMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. 7Muscle GAA was undetectable in most samples, and is therefore not reported. 8Units are expressed relative to wet tissue weight. View Large Clinical chemistry and hematological outcomes were largely unaffected by dietary treatment (Table 7). An interaction (P < 0.05) was observed for glucose, with 0.12% added GAA increasing glucose concentrations by 2.7% in Arg-unsupplemented diets and decreasing glucose concentrations by 4.1% in Arg-supplemented diets. Plasma phosphorus concentrations and glutamic acid (Glu) dehydrogenase activity were both decreased (P < 0.05) due to Arg supplementation, and Glu dehydrogenase activity was also decreased (P < 0.05) due to GAA supplementation. In terms of hematological responses, blood protein concentrations decreased (P < 0.05) due to supplementation of either Arg or GAA (Table 8). As a proportion of total blood leukocytes, heterophils decreased (P < 0.05), while lymphocytes increased (P < 0.05), due to graded GAA supplementation; no other hematological outcomes were affected by dietary treatment. Table 7. Blood clinical pathology outcomes of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Albumin, g/dL  1.10  1.13  1.14  1.1  1.00  1.08  1.05  0.05  0.24  0.56  0.43  Glucose, mg/dL  232.1a,b  244.6b  238.3a,b  241.5a,b  220.4a  231.5a,b  236.8  5.36  0.27  0.73  0.011  Asp aminotransferase, U/L  136.75  148.25  143.88  135  131.75  146.38  145  7.37  0.42  0.46  0.41  Phosphorus, mg/dL  7.60  8.39  7.98  7.53  7.20  7.39  7.68  0.27  0.027  0.70  0.14  Calcium, mg/dL  10.69  10.85  10.68  10.61  10.28  10.85  10.45  0.21  0.50  0.65  0.21  Glu dehydrogenase, U/L  6.78  4.76  5.40  4.95  3.33  3.69  4.81  0.66  0.007  0.025  0.96  Creatine kinase, U/L  2106  1908  2586  2672  2133  2379  2178  304.0  0.62  0.28  0.42    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-values6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Albumin, g/dL  1.10  1.13  1.14  1.1  1.00  1.08  1.05  0.05  0.24  0.56  0.43  Glucose, mg/dL  232.1a,b  244.6b  238.3a,b  241.5a,b  220.4a  231.5a,b  236.8  5.36  0.27  0.73  0.011  Asp aminotransferase, U/L  136.75  148.25  143.88  135  131.75  146.38  145  7.37  0.42  0.46  0.41  Phosphorus, mg/dL  7.60  8.39  7.98  7.53  7.20  7.39  7.68  0.27  0.027  0.70  0.14  Calcium, mg/dL  10.69  10.85  10.68  10.61  10.28  10.85  10.45  0.21  0.50  0.65  0.21  Glu dehydrogenase, U/L  6.78  4.76  5.40  4.95  3.33  3.69  4.81  0.66  0.007  0.025  0.96  Creatine kinase, U/L  2106  1908  2586  2672  2133  2379  2178  304.0  0.62  0.28  0.42  a,bMeans within a row lacking a common superscript letter differ (P < 0.05). *Mean value for this treatment was different from the positive control treatment (P < 0.05). 1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. View Large Table 8. Hematological outcomes of chicks fed Arg-deficient diets.1   Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-Value6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Packed cell volume, %  28.7  28.2  29.1  28.3  28.5  28.9  28.5  0.86  0.99  0.68  0.90  Protein, g/dL  3.36  3.03  3.15  3.18  2.86  2.99  3.03  0.96  0.009  0.003  1.00  Total leukocytes, 103/μL  10.65  12.88  11.08  9.86  9.99  13.97  9.89  1.95  0.97  0.46  0.27  Differential cell proportions, %7   Heterophils  61.0  40.0  35.3  51.0  44.0  37.7  44.0  5.63  0.17  0.001  0.33   Lymphocytes  28.4  44.5  52.2  35.9  40.9  49.6  43.9  6.37  0.27  0.008  0.57   Monocytes  3.67  4.60  2.75  5.13  5.25  5.40  4.71  1.86  0.50  0.88  0.84   Eosinophils  2.80  2.50  1.57  2.57  2.29  2.29  2.25  0.67  0.83  0.36  0.60   Basophils  5.43  8.00  8.38  6.67  7.88  6.57  6.00  1.73  0.84  0.26  0.57    Dietary Treatment2          Suppl. Arg, %  0.00  0.00  0.00  0.16  0.16  0.16  0.32    P-Value6  Variable GAA, %  0.003  0.06  0.12  0.00  0.06  0.12  0.004  SEM5  Arg  GAA  Arg × GAA  Packed cell volume, %  28.7  28.2  29.1  28.3  28.5  28.9  28.5  0.86  0.99  0.68  0.90  Protein, g/dL  3.36  3.03  3.15  3.18  2.86  2.99  3.03  0.96  0.009  0.003  1.00  Total leukocytes, 103/μL  10.65  12.88  11.08  9.86  9.99  13.97  9.89  1.95  0.97  0.46  0.27  Differential cell proportions, %7   Heterophils  61.0  40.0  35.3  51.0  44.0  37.7  44.0  5.63  0.17  0.001  0.33   Lymphocytes  28.4  44.5  52.2  35.9  40.9  49.6  43.9  6.37  0.27  0.008  0.57   Monocytes  3.67  4.60  2.75  5.13  5.25  5.40  4.71  1.86  0.50  0.88  0.84   Eosinophils  2.80  2.50  1.57  2.57  2.29  2.29  2.25  0.67  0.83  0.36  0.60   Basophils  5.43  8.00  8.38  6.67  7.88  6.57  6.00  1.73  0.84  0.26  0.57  1Values are means of 8 replicate pens of 5 chicks during the feeding period 8 to 22 d posthatch. Abbreviations: GAA = guanidinoacetic acid. 2The basal diet was formulated to contain 0.96% total Arg [0.84% standardized ileal digestible (SID) Arg] and was analyzed to contain 0.99% total Arg. 3Negative control treatment. 4Positive control treatment. 5Standard error of the mean that applies to all 7 dietary treatments. 6P-values apply only to the first 6 treatments (excludes positive control treatment) that were included in the 2-way ANOVA. 7Differential cell counts expressed as a proportion of total leukocytes detected. View Large DISCUSSION This study was designed to test the efficacy of GAA for restoring growth performance and muscle phosphagen status in fast-growing broiler chicks fed an Arg-deficient diet based on practical ingredients. Combined with a general lack of effects on hematological and clinical chemistry outcomes, this research provides clear and direct evidence that: 1) Arg deficiency is detrimental to growth performance and produces disruptions in blood and muscle energy metabolites, i.e., Cr and PCr profiles, as well as glycogen levels, and 2) supplementation with Arg and/or GAA ameliorate these effects, though to varying degrees depending on the dietary nutrient. Of particular interest were the findings that Arg and GAA supplementation restored BW gain and G:F parameters elicited by Arg deficiency, as well as robust increases in muscle PCr, both on an absolute basis and relative to the muscle ATP concentration as well as muscle glycogen levels. Improvements in growth performance when GAA was added to Arg-deficient diets are in agreement with previous research conducted by Savage and O’Dell (1960) and Dilger et al. (2013). Whereas there was no BW gain response due to GAA supplementation, this was also in agreement with previous research (Dilger et al., 2013). It should be noted that broilers received a standard starter diet (corn-soybean meal-based) that was adequate in all nutrients, including Arg, prior to initiation of the experimental phase. Acknowledging that there is no ability to store AA, it is possible that pre-testing on an Arg-deficient diet may have altered responses to supplemental Arg and GAA during the 2-week growth period used in our study. Improvements in G:F likely occurred because Arg was spared from serving as a precursor for Cr synthesis (Almquist et al., 1941) and was therefore available for alternative functions throughout the body (e.g., lean tissue accretion); a theory that is in agreement with the research conducted by Edwards et al. (1958). In general, GAA was able to improve, and in some instances restore, growth performance of birds receiving Arg-deficient diets containing practical ingredients common to the U.S. poultry industry. In our study, dietary Arg deficiency decreased concentrations of muscle total Cr, and consequently muscle PCr, compared with the PC diet. Because PCr is used to maintain muscle energy homeostasis (Walker, 1979), the decrease in PCr means that muscles in Arg-deficient birds were less able to maintain energy homeostasis, hence they may have been less tolerable to ATP-consuming processes under stressful conditions (e.g., oxidative stress, disease challenge). Regardless of Arg supplementation level, GAA increased muscle PCr such that they were equal to or greater than those elicited by the PC diet. This finding indicates that GAA improved energy homeostasis in muscles cells when supplemented in an Arg-deficient diet. Relative to the NC diet, total Cr concentrations in tissues were increased due to 0.12% GAA supplementation by 64% or 59% in diets formulated to contain 0.84% or 1.00% SID Arg, respectively. Supplementation of 0.06% GAA elicited significant improvements, primarily when added to diets containing 1.00% SID Arg. These results corroborate recent observations by Michiels et al. (2012), where improvements in Cr and PCr/ATP concentrations in breast meat were reported as a result of graded GAA supplementation of Arg-adequate diets. Because maintenance of PCr is based on Cr being phosphorylated to PCr and exported to sites of ATP usage (Brosnan and Brosnan, 2010; Guimarães-Ferreira, 2014), the increased total Cr concentration may indicate an increased capacity for PCr synthesis. This is confirmed by both the absolute concentration and relative ratio (to ATP) of PCr and total Cr, which were both elevated with GAA supplementation, indicating in both cases that GAA may increase the ability of cells to regenerate ATP more effectively. Because of these increases in muscle metabolite concentrations, and the fact that serum GAA concentrations were responsive to dietary GAA supplementation, we conclude that dietary GAA was successfully absorbed and metabolized to synthesize Cr with efficacy greater than that of dietary Arg, which indicates that GAA supplementation will ultimately spare dietary Arg (Wyss and Kaddurah-Daouk, 2000). Considering the rate-limiting enzyme for Cr synthesis, AGAT, is almost exclusively inhibited by Cr (Walker, 1979), increased production of Cr via dietary GAA supplementation would likely exert Arg-sparing effects by downregulating expression and/or activity of the AGAT enzyme, thereby permitting more dietary Arg to be used for purposes other than Cr synthesis. Arg deficiency was visible from reduced serum concentrations in Arg and elevated serum concentrations of most other essential and non-essential AA. Supplementation of GAA reduced serum AA concentrations in birds, with more drastic decreases observed in diets containing 0.84% SID Arg. This indicates that as the Arg deficiency was alleviated, essential AA concentrations in plasma decreased. Zimmerman and Scott (1965) reported a similar trend in BW gain and plasma AA concentrations as dietary lysine approached the requirement. The improvement in BW gain and decrease in serum AA may be explained in the context of the ideal protein concept, which is based on the premise that optimal performance is limited by availability of the scarcest resource. In these diets, Arg is the most-limiting AA, and as the body has no true AA storage depots, any excess AA remaining after attaining maximal growth are transported via the blood to the liver for deamination and subsequent oxidation. Therefore, GAA added to diets formulated to contain either 0.84% or 1.00% SID Arg may have spared Arg by diminishing the need for Arg to serve as a precursor for Cr synthesis, therefore making more Arg available for incorporation into lean tissue along with excess AA, to ultimately decrease serum AA concentrations. In addition to decreased growth in general, evidence from blood chemistry and hematological outcomes indicate that protein degradation may have occurred in Arg-deficient birds. Serum 3-methylhistidine decreased with dietary supplementation of GAA to Arg-unsupplemented diets, but was unchanged when GAA was added to Arg-supplemented diets. Supplementation of GAA also decreased heterophils as the largest proportion of total leukocytes, while dietary Arg concentration had no effect. Heterophil proportions were reduced with GAA supplementation an average of 35% when birds were supplemented with 0.12% GAA compared with 0.0% GAA. There was also a reciprocal change in lymphocyte percentages, the normal circulating leukocyte (Weiss et al., 2010), as heterophil percentage decreased. The combination of decreased 3-methylhistidine concentrations, total protein and heterophil percentages in birds with increased GAA supplementation indicates that Arg deficiency may have caused minor changes in whole-body protein degradation. Although 3-methylhistidine is an indicator of skeletal protein degradation (Young and Munro, 1978), it is typically related to body weight loss. However, when combined with the high proportion of white blood cells as heterophils, a sign of muscle damage (Weiss et al., 2010), this may suggest greater whole-body protein breakdown occurred in Arg-deficient birds. In this context, GAA supplementation may be seen as having reduced metabolic stress (i.e., protein degradation) caused by dietary Arg deficiency. Alleviation of an Arg deficiency is associated with changes in Arg-related metabolites. Serum ornithine (Orn) concentrations were reduced in birds fed the NC vs. PC diet and increased with Arg supplementation, but GAA supplementation had no effect on this circulating metabolite. The production of Orn may increase with Arg supplementation due to the increased production of urea by avian arginase (Tamir and Ratner, 1963a; Fernandes and Murakami, 2010). Increased serum Orn concentrations may also indicate that more GAA and Orn were formed from Arg and Gly due to Arg supplementation (Walker, 1979). Supplementation of GAA decreased Orn concentrations by at least 33 and 12% in diets formulated to contain 0.84 or 1.00% SID Arg with 0.0% GAA, respectively. This was expected because supplementation of GAA spares dietary Arg to the greatest extent in Arg-deficient diets. Serum citrulline levels were also lower in birds fed NC vs. PC diets, but were increased with supplementation of GAA, although to a much lower effect when dietary Arg was closer to adequate concentrations. This could be due to increased nitric oxide production, which also results in citrulline production (Wu and Morris, 1998), but also a decreased conversion of citrulline to Arg (Tamir and Ratner, 1963b) in chicks under less-deficient conditions. As serum citrulline concentrations were distinctively lower in birds fed NC vs. PC diets, but increased dramatically with GAA supplementation in Arg-unsupplemented diets, serum citrulline levels may be a way to determine Arg deficiency, and thereby validate the ameliorative effect of GAA by sparing Arg in unsupplemented diets. Homocysteine was similar or slightly lower in muscle than in blood, as corroborated by previous findings (Seo, 2005). The markedly lower levels reported previously (9.66, 4.66, and 7.77 nmol/g homocysteine in liver, breast muscle, and blood samples) might be explained by differences in age of the chickens, as we collected samples from 22-d-old birds as compared to the 31-week-old chicken samples analyzed previously. Additionally, Samuels (2003) reported plasma homocysteine concentrations of 35.9 μM in 5-week-old broilers, which are intermediate concentrations to those quantified in our study and those reported by Seo (2005). As opposed to the various ways dietary GAA supplementation affected concentrations of blood AA and other metabolites, it was interesting to note that plasma homocysteine was neither affected by dietary Arg nor GAA supplementation. Considering GAA receives a labile methyl group from S-adenosylmethionine and the fact that homocysteine is a key biomarker for quantifying methylation demand in the body (Brosnan et al., 2004), we conclude that homocysteine was rapidly metabolized by either remethylation or trans-sulfuration (Brosnan et al., 2004). Overall, it can be concluded from this study that dietary GAA may spare Arg when Arg-deficient diets are fed to young broiler chicks. Our results indicate that 0.12% supplemental GAA was capable of ameliorating the effects of an Arg deficiency on growth performance and muscle phosphagen and glycogen concentrations caused by a dietary Arg deficiency, with outcomes due to this treatment closely matching responses of chicks fed an Arg-adequate diet. 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Poultry ScienceOxford University Press

Published: Mar 1, 2018

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