Effect of guanidinoacetic acid supplementation on live performance, meat quality, pectoral myopathies and blood parameters of male broilers fed corn-based diets with or without poultry by-products

Effect of guanidinoacetic acid supplementation on live performance, meat quality, pectoral... ABSTRACT Creatine is a nitrogenous compound naturally occurring in animal tissues and is obtained from dietary animal protein or de novo synthesis from guanidinoacetic acid (GAA). The dietary supply of this semi-essential nutrient could be adversely compromised when feeding purely vegetable-based diets. The objective of this experiment was to evaluate the effects of GAA supplementation in broilers fed corn-based diets with or without the inclusion of poultry by-products (PBP) on live performance, carcass and cut up yields, meat quality, pectoral muscle myopathies, differential blood count, blood clinical chemistry, serum GAA and its metabolites. The treatments consisted of PBP inclusion in the diets at 0 and 5%, with or without GAA supplementation (0 or 0.06%). A total of 1,280 one-d-old male Ross 708 broiler chicks were randomly placed in 64 floor pens with 16 replicates per treatment combination. At 0, 14, 35, 48, and 55 d, pen BW and feed intake were recorded. BW gain and FCR were calculated at the end of each phase. Individual BW was obtained at 55 d and one broiler per pen was selected for blood collection. Additionally, four broilers per pen were selected (including the chicken for blood collection) for processing. Data were analyzed as a randomized complete block design in a 2 × 2 factorial arrangement with PBP and GAA supplementation as main effects. An improvement (P < 0.05) on FCR of 0.019 (g:g) was detected at 55 d due to GAA supplementation. The probability of having breast meat with low severity of wooden breast (score 2) was increased (P < 0.05) by GAA inclusion in diets without PBP. An interaction effect (P < 0.05) was detected on GAA concentration in blood. The supplementation with GAA and PBP inclusion resulted in higher (P < 0.05) GAA serum concentration. Generally, meat quality parameters were not affected by GAA. In conclusion, GAA supplementation improved FCR regardless of dietary PBP and reduced wooden breast severity by increasing score 2 in diets without PBP. INTRODUCTION Guanidinoacetic acid (GAA) is a compound synthesized in the avian kidney and liver (Dilger et al., 2013). The GAA is a precursor of creatine considered to be a more stable molecule compared to creatine (Baker, 2009). Creatine is an important component of the energy delivery process in several tissues, particularly those characterized by a high and/or fluctuating energy demand (Harris et al., 1997). In its phosphorylated form, phosphocreatine, creatine is directly involved in maintaining low adenosine diphosphate concentrations at sites of energy utilization, and in the transfer of high energy phosphate from mitochondria. The highest concentrations of creatine are found in skeletal muscle, which accounts for 95 per cent of the total body pool (Harris et al., 1997). In general, about 1.7% of the creatine and phospho-creatine pool is irreversibly converted to creatinine each day and excreted in the urine (Wyss and Kaddurah-Daouk, 2000). Consequently, creatine must be continually replaced (Lemme et al., 2007). The demand for creatine or its precursors may be supplied either directly from animal protein in the diet or by endogenous synthesis (Wyss and Kaddurah-Daouk, 2000). Creatine is produced naturally in the body from GAA, which in turn is synthesized from the amino acids arginine and glycine (Wyss and Kaddurah-Daouk, 2000). These amino acids are highly involved in muscle metabolism (Lemme et al., 2007). The marked growth-promoting effect of creatine may be most simply explained on an arginine-sparing basis (Dilger et al. 2013; DeGroot, 2014). Arginine and glycine are normally found in higher concentrations in feedstuffs such as animal by-products meal than in corn and soybean meal (Baker, 2009; Li et al., 2011). Poultry by-product meal (PBP) is still one of the most important sources of animal protein in animal feed (Bhargava and O’Neil, 1975; Meeker and Hamilton, 2006). Following the ban for the use of meat and bone meal in feed in 2001 (European Community, 2000), some European poultry producers observed a certain drop in performance (Costrel et al., 2009). This could have been partially caused by insufficient supply of creatine or its precursors Arg and Gly which are supposed to be present in animal derived proteins but absent in vegetable-based feed ingredients (Ringel et al., 2007). Approximately 90% of diets for poultry in the U.S. are comprised by corn and soybean meal (Baker, 2009). Vegetable protein sources have been reported (Gabor et al., 1984; Khan and Cowen, 1977) to have limited (< 0.01 mg/g) or no creatine content. However, animal by-products ingredient may have higher amounts of this nutrient depending on the type of manufacturing and quality (Harris et al., 1997; Dobenecker and Braun, 2015). In general, poultry by-products may contain low levels of creatine (Dobenecker and Braun, 2015), but 3.25 times more Gly and 32% more Arg than soybean meal (AminoDat 5.0). Consequently, broilers fed vegetable-based diets without animal by-products may face the possibility of lower supply of Arg and Gly for creatine endogenous production affecting muscle development. According to Petracci et al. (2015), novel pectoral myopathies, such as white striping (WS) and wooden breast (WB) are characterized by a reduced supply of oxygen and nutrients to muscle cell. In addition, this could also be associated with the inadequate elimination of metabolic intermediate products, leading to a membrane dysfunction and increased plasma concentration of enzymes such as creatine kinase (CK) and lactate dehydrogenase (LDH) (Mitchell, 1999; Sandercock and Mitchell, 2003; MacRae et al., 2006). Several management and nutritional solutions have been proposed to minimize and solve these myopathies and meat quality abnormalities. Among these strategies, the reduction of growth rate by using feed restriction programs, reduction of amino acids, lowering the energy value of the diets (Kuttappan et al., 2012a; Trocino et al., 2015; Radaelli et al., 2017), and higher dietary vitamin E and selenium (Guetchom et al., 2012; Kuttappan et al., 2012b) had been tested showing some benefits, improving meat quality and reducing occurrence of myopathies. However, the reduction of growth rate could depress productivity in broiler operations. Based on mice trials, it has been proposed that creatine may have a protective effect on certain neuromuscular (Tarnopolsky, 2007; Chung et al., 2007), and neuro-degenerative (Bender et al., 2006; Kolling and Wyse, 2010; Beal, 2011) diseases, and it also may reverse muscular dystrophy (Nabuurs et al., 2013). These results may be due to the capacity of creatine to prevent the inhibition of energy metabolism and lipid peroxidation. Therefore, our study evaluated a possible beneficial effect of GAA supplementation on pectoral myopathies without reducing the genetic potential for growth of broilers. Creatine production is a very important muscle metabolite and the introduction of an additive like GAA for creatine synthesis may influence blood markers related to broiler health and energy utilization and metabolism. Available published data regarding blood chemistry to assess metabolic status of heavy broilers fed PBP, and GAA, and compared with purely vegetable-based diets have not been reported so far. In the U.S., 47.5% of broilers are grown for more than 56 d (Agristats, 2016), therefore the present experiment focused on metabolites and breast meat quality parameters in heavy broilers of eight wk of age. Previous studies showed that GAA supplementation lowered breast meat pH, affecting the water holding capacity and improving the meat tenderness. Kuttappan et al. (2017) reported that WB and WS pectoral myopathies are highly correlated with meat quality parameters such us higher ultimate pH (>6.04), and drip loss. Considering that it has been reported that dietary GAA supplementation slightly affected ultimate pH in diets without fish meal (Michiels et al., 2012), there could be a possible beneficial effect on the occurrence of pectoral myopathies. Previous experiments regarding GAA supplementation did not evaluate the effects of dietary GAA inclusion on pectoral myopathies. Consequently, the objective of this study was to evaluate the effects of GAA supplementation on live performance, meat quality, metabolite serum concentrations, blood hematological profile and chemistry, and pectoral myopathies severity in heavy broilers fed corn-soybean meal based diets with or without PBP. MATERIALS AND METHODS Treatments and Birds Husbandry All the procedures involving the birds used in the present experiment were approved by the North Carolina State University Institutional Animal Care and Use Committee. Four dietary treatments from a 2 × 2 factorial arrangement with PBP and GAA as main effects were considered. Diets contained two levels of poultry by-products (0 vs. 5%), and two levels of GAA (0 and 0.06%). The study herein was conducted in an open-sided house with clear curtains and negative pressure ventilation. A total of 1,280 Ross-708-d-old male chicks were randomly placed in 64 floor pens (3.81 × 1.19 m) 16 pens per treatment and 20 chicks per pen (4.42 broilers/m2 at placement) for a final stocking density of 23.2 kg/m2 at 55d of age. Used litter was employed as bedding in the pens and broilers were exposed to continuous light on a 23L:1D (30 lux light intensity) program during the first seven d of age. Day length was then gradually reduced to 17L:7D (10 lux) up to 28 d of age. From 28 d until the end of the experiment, light program was maintained at 17L:7D with an intensity of 5 lux. At this age, natural daylight length lasted from 6:30 am to 5:30 pm, and supplemental light was offered from 5:30 pm to 9:00 pm. Brooding house temperature was set at 33.6°C at placement and gradually reduced until 20.6°C at 21 d and kept until d 56 to guarantee chicken environmental comfort. Diets Dietary treatments were obtained from two basal diets to ensure similar nutrient content among treatments in all experimental phases. Basal feeds consisted in corn-soybean meal based diets with or without the inclusion of PBP (0 vs. 5%). Subsequently, GAA was supplemented either 0 or 600 g of GAA per ton of feed (recommended dose, European Commission) in the form of CreAMINO® (Evonik Nutrition & Care Gmbh, Hanau, Germany) according to the corresponding treatment (Table 1). Diets that did not contain GAA had 600 g of sand per ton of feed. All dietary treatments were formulated to be isoenergetic, isonitrogenous, and mixed to represent typical U.S. broiler industry practices (AgriStats, 2016). Formulated digestible amino acid levels were based on AminoDat® 5.0 (2015) recommendations (Table 2). Macro ingredients (corn, soybean meal, and distilled dried grain with solubles) were analyzed for total amino acid and ME content prior to diet formulation. Digestible amino acid content was calculated from the total amino acid content obtained from lab analyses and using table values for digestibility coefficients (AminoDat 5.0, 2015). The ME values (kcal/kg) were obtained from an in vivo trial with roosters (Dr. Nick Dale, University of Georgia). Total and digestible amino acid content for PBP was obtained using the same procedures as described for the macro ingredients. Creatine-concentration in PBP diets were determined by AlzChem AG (Trosberg, Germany) according to the ion chromatography method (CRL Feed Additives, 2007). Starter, grower, finisher and withdrawal diets were fed from 0–14, 15–35, 36–42, and 43–56 d of age, respectively. Starter was fed in crumbles and all other diets in pellets. Table 1. Ingredient composition of starter, grower, finisher and withdrawal basal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00  1Coban® 90 (Monensin), Elanco Animal Health, Greenfield, IN, at 500 g/ton in the starter and grower diets. 2Trace minerals provided per kg of premix: manganese (Mn SO4), 60 g; zinc (ZnSO4), 60 g; iron (FeSO4), 40 g; copper (CuSO4), 5 g; iodine (Ca(IO3)2),1.25 g. 3Vitamins provided per kg of premix: vitamin A, 13,227,513 IU; vitamin D3, 3,968,253 IU; vitamin E, 66,137 IU; vitamin B12, 39.6 mg; riboflavin, 13,227 mg; niacin, 110,229 mg; d-pantothenic acid, 22,045 mg; menadione, 3,968 mg; folic acid, 2,204 mg; vitamin B6, 7,936 mg; thiamine, 3,968 mg; biotin, 253.5 mg. 4Quantum Blue 5G® at 0.176 lbs/ton (80 g/ton) to provide 500 FYT (AB Vista) delivering 0.13% of available P, 0.06% of calcium and 0.03% of sodium. 5CreAMINO®: Guanidinoacetic acid (GAA) with 96% of concentration, Lot numbers: 3/29/16, 9/26/16. View Large Table 1. Ingredient composition of starter, grower, finisher and withdrawal basal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00  1Coban® 90 (Monensin), Elanco Animal Health, Greenfield, IN, at 500 g/ton in the starter and grower diets. 2Trace minerals provided per kg of premix: manganese (Mn SO4), 60 g; zinc (ZnSO4), 60 g; iron (FeSO4), 40 g; copper (CuSO4), 5 g; iodine (Ca(IO3)2),1.25 g. 3Vitamins provided per kg of premix: vitamin A, 13,227,513 IU; vitamin D3, 3,968,253 IU; vitamin E, 66,137 IU; vitamin B12, 39.6 mg; riboflavin, 13,227 mg; niacin, 110,229 mg; d-pantothenic acid, 22,045 mg; menadione, 3,968 mg; folic acid, 2,204 mg; vitamin B6, 7,936 mg; thiamine, 3,968 mg; biotin, 253.5 mg. 4Quantum Blue 5G® at 0.176 lbs/ton (80 g/ton) to provide 500 FYT (AB Vista) delivering 0.13% of available P, 0.06% of calcium and 0.03% of sodium. 5CreAMINO®: Guanidinoacetic acid (GAA) with 96% of concentration, Lot numbers: 3/29/16, 9/26/16. View Large Table 2. Calculated and analyzed nutrient content of basal starter, grower, finisher, and withdrawal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18  1Analyzed values are means of 2 samples. Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large Table 2. Calculated and analyzed nutrient content of basal starter, grower, finisher, and withdrawal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18  1Analyzed values are means of 2 samples. Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large For the pelleting process, a temperature between 82 and 85°C in the conditioner was used for 30 seconds. The steam pressure was 32 psi (221 kPa), and the pellet die was 11/64″ x 1″ 3/8″ (4.4 × 34.9 mm) for an L/D ratio of 8. The capacity of pelleting used was 2 to 5 ton/hour (2,000 to 5,000 kg per hour) to improve pellet quality. Representative samples of each final dietary treatment were collected after crumbling (starter) or pelleting process, to analyze creatine, and GAA content according to the IC method (CRL Feed Additives, 2007), and fully validated procedures (Dobenecker and Braun, 2015) by AlzChem AG, Trostberg, Germany (Table 3). For each one of the dietary phases 0.85, 2.90, and 2.48 kg of starter, grower and finisher, respectively, were offered for each bird alive during each phase. The withdrawal diet was offered ad libitum. Water was provided for ad libitum consumption. Feeders were shaken twice daily to stimulate uniform feed intake. Table 3. Calculated and analyzed creatine and GAA content of the dietary treatments for Ross-708 male broilers. Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  1Values are means of 2 samples. Report issued by the Alzchem AG according to the IC method (CRL Feed Additives, 2007) 2Calculated values (CreAMINO®, GAA min. 96%), Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large Table 3. Calculated and analyzed creatine and GAA content of the dietary treatments for Ross-708 male broilers. Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  1Values are means of 2 samples. Report issued by the Alzchem AG according to the IC method (CRL Feed Additives, 2007) 2Calculated values (CreAMINO®, GAA min. 96%), Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large Live Performance and Blood Collection Group BW was obtained at 0, 14, 35, 48, and 55 d of age. Feed intake, BW gain, and feed conversion ratio (FCR) were calculated at the end of each phase. Mortality was monitored and recorded twice daily. At 55 d, individual BW were obtained to calculate flock uniformity using the CV%. One broiler per pen was randomly selected to collect blood samples from non-fasting chickens. These samples were obtained from the brachial vein into plain, heparinized or EDTA containing tubes for hematologic profile with differential for white blood cells count (WBC), red blood cells count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), count of heterophils, thrombocytes, monocytes, lymphocytes, T-cell, B-cell. Differential blood count analysis (thrombocytes, heterophils, monocytes, lymphocytes, T-cells, and B-cells) was performed by LMU Ludwig-Maximilians University Laboratory in Munich, Germany, as described by Seliger et al. (2012) using a BD FACS Canto II instrument (Becton Dickinson, Heidelberg, Germany) for cell counting and BD FACS DIVA and FlowJo (Tree Star Inc., OR, USA) software for data analysis. For clinical hematology analysis and red blood cell count, samples were submitted to a commercial laboratory (Synlab Vet Laboratory, Cologne, Germany), to analyze erythrocytes, hemoglobin, hematocrit, MCV, MCH, and MCHC, using flow cytometry (FCM), and ADVIA 2120 analyzer from Siemens (Siemens, Deutschland GmbH, Hamburg, Germany). Serum enzyme activity of creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and lactate dehydrogenase (LDH) were analyzed, as well as glucose, uric acid, minerals (phosphorus, calcium, sodium, potassium, and chloride), total protein, albumin, and cholesterol were also analyzed at North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA). Serum [creatine, creatinine, and GAA] and homocysteine in plasma were analyzed using standardized procedures (Baylor University, Houston, TX). Selection of Birds for Processing At 55 d, individual and group BW were obtained, and the average for each pen was calculated. Four broilers (including the selected for blood collection) per pen were selected with BW within two standard deviations above or under their corresponding average for each pen. Carcass and Cut up Yields At 56 d, feed was withdrawn for 12-h. Broilers were slaughtered at the NCSU pilot processing plant. Broilers were weighed, electrically stunned for 11 s, killed by exsanguination, and allowed to bleed for 90 s. Broilers were then scalded at 55°C for 90 s, picked for 30 s, and manually eviscerated. Carcasses were dressed by removing liver, gizzard, heart, oil gland, crop, proventriculus, lungs, and viscera. Carcasses were then air-chilled for six h, and manually deboned on stationary cones. Parts of the leg quarters, breast fillets (Pectoralis major), breast tenders (Pectoralis minor), wings, and rack with skin were obtained and weighed. The carcass yield was calculated for the chilled carcass as a percentage of the fasted live BW. Cut up yields were expressed as a percentage of the chilled absolute carcass BW. Meat Quality Evaluation To determine drip loss, the right Pectoralis major muscle was weighed six h post-mortem and immediately placed in a plastic bag, hung from a hook, and stored between 4–6°C for 24 h. After hanging, the sample was gently wiped with paper and weighed again. The difference in weight corresponded to the drip loss and was expressed as the percentage of the initial muscle weight. For cook loss, the left breast fillets (Pectoralis major) were weighed 24 h postmortem, placed on grilled-aluminum trays, and cooked in a forced air oven (SilverStar Southbend, Model SLES/10sc, gas type, NC, USA). Fillets were cooked to an internal temperature of 75°C (approximately 35 min), as measured by a Therma Plus thermocouple with a 10-cm needle temperature probe (ThermoWorks Model 221-071, UT, USA). The cooked fillets were cooled to room temperature, wiped gently with paper and re-weighed to determine cook yield as a percentage of the cooked weight relative to the raw weight. Shear force (kg) of cooked breast fillets samples was determined using a Warner-Bratzler shear device (Warner-Bratzler meat shear, Bodine Electric Company, Chicago, USA). Two samples per breast fillets (2 × 2 × 2 cm3) were sheared in a direction perpendicular to the muscle fibers. The maximum force measured when cutting the samples was expressed in kg force. Postmortem pH (t = 1, 6 and 24 h) was measured after skin was removed from Pectoralis major muscle samples using a portable pH meter (Oakton waterproof pH Tester 30). Color was measured on skinless Pectoralis major samples by the CIE L* (lightness), a* (redness), and b* (yellowness) system using a Minolta Chroma Meter CR-400 (Konica Minolta Sensing, Inc., Japan). A measuring area of 10 mm and illuminant D65 and 2° standard observer were used. The colorimeter was calibrated using a white tile (reference number 13033071; Y = 93.9, x = 0.3156, y = 0.3318). Pectoral Myopathies Skinless Pectoralis major muscle samples were visually evaluated to determine pectoral myopathies (spaghetti muscle, WB and WS) severity. The evaluation was performed by two experts in the field (College of Veterinary Medicine, North Carolina State University). Spaghetti muscle was recorded as presence or absence of this abnormality, whereas WS and WB were scored based on severity. The WB severity was based on a four-point scale as described by Tijare et al. (2016). Fillets samples were manually palpated and determined a score of 1 as a normal fillet with no WB signs, score 2 was considered a low severity, score 3 a medium, and score 4 as severe. The WS severity was scored with the scale used by Kuttappan et al. (2012c) which considered a three-point based scale of severity. Score 1 described a breast fillet with no white striations on the surface. Score 2 were the fillets with white striations less than 1 mm of thickness and easily observed in the surface, and score 3 was represented by the white striation more than 1 mm of thickness. In addition, the probability distribution for myopathies (WB and WS) scores was analyzed. Statistical Analysis Data were analyzed as completely randomized block design with a 2 × 2 factorial arrangement of treatments with the inclusion of PBP (0 vs. 5%), and GAA supplementation (0 vs. 0.06%) as main effects to have a total of four treatments. Each treatment respectively had 16 replicates distributed equally in four blocks (location of pens within the house) that were considered random effect. Data was analyzed in JMP 12 (SAS Institute. Inc., Cary, NC, 2016) using ANOVA in a mixed model. Differences between means were separated by the LS means method using Tukey's or t-student test at a level of significance of alpha = 0.05. Additional to this, for blood, carcass and cut up yields, meat quality and pectoral myopathies results, individual broiler's data from the same pen were nested inside every corresponding treatment and considered as random effect. Cutter was also included in the model as random effect for carcass and cut up yields. Data of scores probability distribution for WB and WS were analyzed using GLIMMIX (SAS Institute, 2008). RESULTS AND DISCUSSION Diets and Live Performance The analysis of GAA, and creatine are shown in Table 3. The GAA results showed slight differences on concentrations compared to the intended dose (600 mg/kg). The concentration of CreAMINO® in feed was calculated according to the standard concentration of GAA (CreAMINO®, GAA, 96% min, AlzChem AG, Trostberg, Germany) in the product. As expected, low or absence of creatine was obtained in all the dietary treatments (Table 3). Creatine analysis of PBP indicated a concentration of 156 mg/kg that did not affect final dietary concentration of creatine. Analyzed total amino acid and crude protein content were similar to formulated values and these are presented in Table 2. No interaction (P > 0.05) effects of treatments were observed on BW, BW gain, feed intake, and FCR in any of the phases evaluated (Table 4). Feed intake was not affected (P > 0.05) by PBP inclusion in the diet nor GAA addition throughout the whole experiment. At 0–14, 0–48 and 0–55 d, dietary inclusion of PBP decreased (P < 0.05) BW and BW gain. Broilers fed diets without PBP were heavier and gained more weight than broilers that were fed diets with PBP. The FCR was not affected (P > 0.05) by PBP or GAA at 0–14d (data not shown) and 0–35 d. However, at 0–48 and 0–55 d, FCR was improved (P < 0.01) up to two points by GAA supplementation regardless of the inclusion of PBP in the diet. At these ages, broilers fed diets with PBP had worse (P < 0.05) FCR than broilers fed diets without PBP. Diets without PBP and with GAA supplementation had slightly worse (P < 0.05) flock uniformity with higher CV% of individual BW. Total mortality was not affected (P > 0.05) by PBP inclusion or GAA supplementation throughout the experiment with no mortality in some pens. In general, overall average mortality rates per pen was close to 8% up to 55d including culled birds due to leg problems. Table 4. Effect of supplementation of guanidinoacetic acid (GAA) in corn-based diets with or without poultry by-products for Ross-708 male broilers on live performance up to 55 d of age.1 Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  1Values are means of 16 pens per treatment combination with 20 male broiler chickens. 2Flock uniformity at 55 d of age. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Table 4. Effect of supplementation of guanidinoacetic acid (GAA) in corn-based diets with or without poultry by-products for Ross-708 male broilers on live performance up to 55 d of age.1 Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  1Values are means of 16 pens per treatment combination with 20 male broiler chickens. 2Flock uniformity at 55 d of age. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Previous studies (Escalona and Pesti, 1987; Caires et al., 2010) concluded that PBP dietary inclusion did not affect live performance when including up to 5% in broiler diets. In the present experiment, the dietary inclusion of PBP resulted in reduced live performance. This result was unexpected, since the physical evaluation and laboratory analyses of nutrient content resulted in normal bromatological characteristics of this feed ingredient with no microbiological contamination. A possible over-estimation of digestible amino acid content in this feed ingredient may explain the observed response on live performance. Moreover, results reported by Michiels et al. (2012) suggested that no improvements on broiler live performance were observed when GAA supplemented diets were compared with a positive control diet containing fish meal. Conversely, Lemme et al (2011) concluded that dietary GAA improved live performance in diets containing fish meal. The present study confirmed that GAA at a level of 0.06% can effectively improve FCR and BW gain in diets containing animal meals, in this case PBP. Previous research validated the efficacy of dietary GAA inclusion on improving live performance in chickens that were fed diets containing only vegetable ingredients (Lemme et al., 2007; Ringel et al., 2007; Costrel et al., 2009; Michiels et al., 2012; DeGroot, 2014), as well as in arginine-deficient diets in young chicks due to the sparing effect of GAA on Arg (Savage and O΄|'Dell, 1960; Dilger et al., 2013; DeGroot, 2014; DeGroot et al., 2015). Therefore, dietary GAA supplementation improved live performance even in cases where Arg supply in feed was deficient. Results observed in the present study suggested that dietary GAA improved live performance regardless of the inclusion of PBP in the diet. Carcass Yield and Cut up Parts Dietary PBP and GAA showed interaction effects (P < 0.05) on Pectoralis major and breast meat yield. GAA supplementation improved Pectoralis major and breast meat yield only in diets containing PBP (Table 5). Broilers fed diets containing PBP and supplemented with GAA had higher (P < 0.05) yields compared to broilers fed non-supplemented diets with PBP. Carcass, wings, and leg quarters yields were not affected (P > 0.05) either by dietary PBP or GAA. Caires et al. (2010) observed no differences (P > 0.05) on carcass and breast meat yields when corn-soybean meal diets were compared to diets containing PBP (5%). Michiels et al. (2012) observed improvements (P < 0.05) on breast meat yield by up to 1.04-fold when GAA was added (0.06% and 1.2%) in corn-based diets. However, when treatments were compared with a positive control containing fish meal, this effect was not observed (P > 0.05). Interestingly, in the current trial the dietary GAA supplementation improved breast meat yield only in diets containing PBP. Table 5. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross-708 male broilers on carcass, breast meat, and cut up part yields at 56 d of age.1         Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602          Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602  1Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment and cutter (random effects) 2Relative to live weight of broilers selected for processing. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Table 5. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross-708 male broilers on carcass, breast meat, and cut up part yields at 56 d of age.1         Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602          Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602  1Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment and cutter (random effects) 2Relative to live weight of broilers selected for processing. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Meat Quality Generally, meat quality was not affected either (P > 0.05) by dietary PBP or GAA, except for an interaction effect (P < 0.05) observed on cook loss, and an effect on the b* (yellowness) value due to PBP inclusion in the diet. Broilers fed diets with PBP and not supplemented with GAA had the lowest (P < 0.05) cook loss (Table 6). Broilers fed diets with PBP had higher (P < 0.05) b* value than the ones fed diets without PBP (10.06 vs. 9.27). Table 6. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products on meat quality parameters at 56 d of age for Ross-708 male broilers.* Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  *Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment (random effects) a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. 1Warner-Bratzler; Color: L* = lightness, a* = redness, b* = yellowness 2(3 = severe; 2 = medium; 1 = low) 3(4 = severe; 3 = medium; 2 = low; 1 = normal) View Large Table 6. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products on meat quality parameters at 56 d of age for Ross-708 male broilers.* Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  *Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment (random effects) a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. 1Warner-Bratzler; Color: L* = lightness, a* = redness, b* = yellowness 2(3 = severe; 2 = medium; 1 = low) 3(4 = severe; 3 = medium; 2 = low; 1 = normal) View Large Fletcher et al. (2000) concluded that the ultimate pH of normal chicken breast meat ranges between 5.7 and 5.96. No differences (P > 0.05) due to treatments were observed in the present experiment and results indicated that ultimate pH ranged from 5.99 to 6.03. Michiels et al. (2012) also did not observed effects of GAA supplementation on postmortem pH, shear force, and a* value in diets containing fish meal. Generally, our measurements of shear force (Warner-Bratzler) ranged from 42.2 to 43.8 N (4.30 to 4.47 kg), which are values that can be considered as “moderately to slightly tender” category based on Lyon and Lyon (1991) sensory ranges. In addition, Poole et al. (1999) found that broilers around 56 d of age should have an average value of shear force (Warner-Bratzler) of about 4.64 kg ± 0.18 kg. They suggested that breast fillets in the scale from 3.46 to 6.41 kg are considered “moderate tender” which are similar values to the ones observed in the current experiment. Fletcher (2002) reported that differences in tenderness depend on age of the chickens and can be due to the fact that heavier broilers are more mature at the time of harvest and have more cross-linking of collagen. Therefore, results of our experiment suggested that the values of shear force obtained were related to age of the chickens at processing. Results obtained in b* value of the study herein could be explained by the level of inclusion of corn in the diet, and the theoretically carotenoid content in PBP (Sajilata et al., 2008). All experimental diets containing PBP had higher inclusion of corn, compared to diets that did not contain PBP throughout the whole experiment. Consequently, they contained a greater content of carotenoids in the feed that might have been deposited later in breast muscle. Pectoral Myopathies The occurrence (presence or absence) of “spaghetti muscle” was evaluated, but not enough breast fillets were affected with this myopathy to have enough replicates for statistical analysis. The WS and WB overall average scores were not affected (P > 0.05) either by dietary PBP inclusion or GAA supplementation (Table 6). However, an interaction effect (P < 0.05) was detected on the probability score distribution (Figure 1) for WB score 2 (low severity). In diets without PBP, the supplementation with GAA increased the probability of having WB score 2 up to two fold compared to samples from broilers fed non supplemented diets. This response is explained mainly by the reduction of score 3 due to GAA supplementation in diets without PBP. Additionally, in diets containing PBP the probability for WB score 2 was not different between treatments. No effects (P > 0.05) due to PBP inclusion or GAA supplementation were observed on the probability distribution for WS myopathy. Figure 1. View largeDownload slide Interaction effect of dietary PBP and GAA supplementation on probability distribution (0.00 – 1.00) for each wooden breast severity score in Ross-708 male broilers at 56 d of age. Means not sharing a common superscript (a-b) are significantly different (P < 0.05) by Tukey's test. Each value represents the probability (0.0 – 1.0) of developing each severity score according to main factors or factorial arrangement of treatments, n = 52 within 16 replicates per treatment combination. Scores are based on a 4-point scale (4 = severe, 3 = medium, 2 = low, 1 = normal). Figure 1. View largeDownload slide Interaction effect of dietary PBP and GAA supplementation on probability distribution (0.00 – 1.00) for each wooden breast severity score in Ross-708 male broilers at 56 d of age. Means not sharing a common superscript (a-b) are significantly different (P < 0.05) by Tukey's test. Each value represents the probability (0.0 – 1.0) of developing each severity score according to main factors or factorial arrangement of treatments, n = 52 within 16 replicates per treatment combination. Scores are based on a 4-point scale (4 = severe, 3 = medium, 2 = low, 1 = normal). According to Kuttappan et al. (2017), pH of breast meat affected with either WS, WB or both, presented higher (>6.04), ultimate pH values compared to samples without signs of these abnormalities. Khan (1974) concluded that meat pH is determined by muscle glycogen content and its degradation rate. Mudalal et al. (2014) suggested that GAA supplementation could avoid glycogen depletion and improve meat quality. Recently, Majdeddin et al. (2017) showed that higher phosphocreatine, creatine and glycogen concentrations in breast muscle of broilers were observed with supplementation of GAA (0.06 and 0.12%). Consequently, our results on WB may be a response driven by phosphocreatine and creatine more than by glycogen since no changes on ultimate pH were observed. Previous studies (Ringel et al., 2007; Michiels et al., 2012; DeGroot, 2014) showed that GAA supplementation increased the concentration of creatine in breast muscle. Intramuscular phospho-creatine can attract water into the muscle cell and increase the cell volume (Hultman et al., 1996). Haussinger (1996) found that a super-hydrated muscle may trigger protein synthesis, minimize protein breakdown, and increase glycogen synthesis. Our results may be explained by the protective effects of creatine found in muscle energy metabolism as reported in previous studies in humans (Balsom et al., 1994), rats (Kolling and Wyse, 2010; Kolling et al., 2013; Nabuurs et al., 2013) with muscle dystrophies (Pearlman and Fielding, 2006; Chung et al., 2007; Tarnopolsky, 2007). Nabuurs et al. (2013) concluded that muscular dystrophy could be reversed in rats by dietary supplementation of creatine. Kolling et al. (2013) suggested that the effect of creatine and its interaction with homocysteine altered glucose oxidation and protected muscle from energy imbalances in rats. It has been demonstrated that alterations in energy metabolism seem to be implicated in the pathogenesis of several muscle and neurological complications, metabolic disorders, aging and neuromuscular diseases including WB in broilers (Abasht et al., 2016). Therefore, the supplementation with GAA as a precursor of creatine could reduce the WB severity by supplying enough creatine avoiding glycogen depletion and consequently preventing muscle damage. Blood Analysis Blood results (Table 7) showed interaction effects (P < 0.05) of treatments only on GAA (Table 8) and MCV (Table 9) serum concentration. Broilers fed diets containing PBP without GAA supplementation had the lowest (P < 0.05) MCV serum concentration. Serum GAA concentration of broilers fed diets with PBP and GAA addition were 12.4 times higher (P < 0.05) than chickens fed non-supplemented diets. Broilers fed diets without PBP and GAA dietary supplementation increased the blood GAA up to 10 times compared to chickens fed non-supplemented diets. These results confirmed effective GAA uptake and metabolism independently of PBP inclusion. Hematological results (Table 9) of blood cells count were not affected (P > 0.05) by PBP inclusion nor GAA supplementation. Serum cholesterol concentration (Table 7) in broilers fed diets with PBP (140.34 mg/dl) was higher (P = 0.05) than chickens fed diets without PBP (133.15 mg/dl). On the other hand, other blood (Tables 7, 8, and 9) parameters (albumin, protein, glucose, uric acid, HBE, MCH, homocysteine), minerals (P, Ca, Na, K, Cl), and hepatic enzymes (ALT, AST, GGT, LD, CK) were not affected (P > 0.05) either by PBP inclusion in the diet or GAA supplementation. However, trends presented in Tables 7, 8 and 9 were observed on serum concentration of glucose (P = 0.06), chloride (P = 0.079), erythrocytes (P = 0.058), and hematocrit (P = 0.063) due to dietary PBP inclusion, and a trend in GGT (P = 0.069) attributed to GAA supplementation was found. Creatine serum concentration was higher (P < 0.01) in broilers fed supplemented diets than chickens fed non-supplemented diets (65.87 vs. 41.46 μM) due to dietary GAA. Moreover, broilers fed diets containing PBP had higher (P < 0.01) serum creatinine concentration compared to broilers fed diets without PBP (0.79 vs. 0.36 μg/ml). Table 7. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum metabolites at 55 d.1 Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Analysis were performed at North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA). View Large Table 7. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum metabolites at 55 d.1 Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Analysis were performed at North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA). View Large Table 8. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum liver and muscle enzymes and metabolites at 55 d.1 Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a–cMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (gamma-glutamyl transpeptidase), LDH (lactic acid dehydrogenase), CK (creatine kinase). Analysis were performed by North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA), and Baylor University, Houston, TX. View Large Table 8. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum liver and muscle enzymes and metabolites at 55 d.1 Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a–cMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (gamma-glutamyl transpeptidase), LDH (lactic acid dehydrogenase), CK (creatine kinase). Analysis were performed by North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA), and Baylor University, Houston, TX. View Large Table 9. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on hematological parameters at 55 d.1 Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration). 2Flow cytometry method. 3Results calculated. LMU Ludwig-Maximilians University Laboratory in Munich, Germany, and Synlab Vet Laboratory, Cologne, Germany. View Large Table 9. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on hematological parameters at 55 d.1 Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration). 2Flow cytometry method. 3Results calculated. LMU Ludwig-Maximilians University Laboratory in Munich, Germany, and Synlab Vet Laboratory, Cologne, Germany. View Large Zhang et al. (2017) reported that certain values in hematology may be affected by dietary GAA depending on dose and diet composition. DeGroot (2014) found no effects on blood cell count (leukocytes, heterophils, lymphocytes, monocytes, eosinophils, basophils), and only changes on differential cell proportions in heterophils and lymphocytes due to GAA supplementation in broilers fed both arginine-deficient and arginine-adequate diets. Previous trials (Tossenberger et al., 2016) showed that serum protein, albumin, cholesterol, glucose, urea, uric acid and enzymes ALT, AST, and GGT remained essentially unchanged (P > 0.05) even when dietary GAA inclusion was up to 0.6%. Likewise, Michiels et al. (2012) found no differences on serum glucose and uric acid in broilers fed corn-based diets without fish-meal and raised up to 26 d. According to DeGroot (2014), no differences (P > 0.05) attributed to GAA supplementation were detected on AST, CK and mineral (Ca, P) serum concentrations. In the experiment here in, the supplementation with GAA only increased GAA and creatine serum concentrations. Consequently, our results are in agreement with previous reports. Abasht et al. (2016) reported higher levels of gamma-glutamyl amino acid catabolite 5-oxoproline in tissue of samples affected by WB up to 1.57-fold as compared to unaffected samples. This suggests the import of extracellular glutathione that together with other biomarkers indicated muscle degradation and oxidative stress. Considering our findings, we may hypothesize that GAA reduced WB severity and the lower GGT serum blood level is related to this effect, despite of no effects on CK. Similar to our findings, DeGroot (2014) found that dietary GAA increased (P < 0.01) creatine and GAA serum concentration. Petracci et al. (2015) discussed that the presence of pectoral myopathies may be related by an increased concentration of CK and LDH. Although, reductions in WB severity were observed in the present experiment, no effects on serum concentration of these enzymes were detected due to GAA supplementation or PBP inclusion. This study showed generally no difference in endpoints determined in blood other than GAA and creatine which is expected. Furthermore, our findings provide evidence that dietary GAA supplementation improved broiler live performance up to 55 d, independently of PBP inclusion in the diets. 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Nutr.  29: 456– 464. © 2018 Poultry Science Association Inc. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Effect of guanidinoacetic acid supplementation on live performance, meat quality, pectoral myopathies and blood parameters of male broilers fed corn-based diets with or without poultry by-products

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey097
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Abstract

ABSTRACT Creatine is a nitrogenous compound naturally occurring in animal tissues and is obtained from dietary animal protein or de novo synthesis from guanidinoacetic acid (GAA). The dietary supply of this semi-essential nutrient could be adversely compromised when feeding purely vegetable-based diets. The objective of this experiment was to evaluate the effects of GAA supplementation in broilers fed corn-based diets with or without the inclusion of poultry by-products (PBP) on live performance, carcass and cut up yields, meat quality, pectoral muscle myopathies, differential blood count, blood clinical chemistry, serum GAA and its metabolites. The treatments consisted of PBP inclusion in the diets at 0 and 5%, with or without GAA supplementation (0 or 0.06%). A total of 1,280 one-d-old male Ross 708 broiler chicks were randomly placed in 64 floor pens with 16 replicates per treatment combination. At 0, 14, 35, 48, and 55 d, pen BW and feed intake were recorded. BW gain and FCR were calculated at the end of each phase. Individual BW was obtained at 55 d and one broiler per pen was selected for blood collection. Additionally, four broilers per pen were selected (including the chicken for blood collection) for processing. Data were analyzed as a randomized complete block design in a 2 × 2 factorial arrangement with PBP and GAA supplementation as main effects. An improvement (P < 0.05) on FCR of 0.019 (g:g) was detected at 55 d due to GAA supplementation. The probability of having breast meat with low severity of wooden breast (score 2) was increased (P < 0.05) by GAA inclusion in diets without PBP. An interaction effect (P < 0.05) was detected on GAA concentration in blood. The supplementation with GAA and PBP inclusion resulted in higher (P < 0.05) GAA serum concentration. Generally, meat quality parameters were not affected by GAA. In conclusion, GAA supplementation improved FCR regardless of dietary PBP and reduced wooden breast severity by increasing score 2 in diets without PBP. INTRODUCTION Guanidinoacetic acid (GAA) is a compound synthesized in the avian kidney and liver (Dilger et al., 2013). The GAA is a precursor of creatine considered to be a more stable molecule compared to creatine (Baker, 2009). Creatine is an important component of the energy delivery process in several tissues, particularly those characterized by a high and/or fluctuating energy demand (Harris et al., 1997). In its phosphorylated form, phosphocreatine, creatine is directly involved in maintaining low adenosine diphosphate concentrations at sites of energy utilization, and in the transfer of high energy phosphate from mitochondria. The highest concentrations of creatine are found in skeletal muscle, which accounts for 95 per cent of the total body pool (Harris et al., 1997). In general, about 1.7% of the creatine and phospho-creatine pool is irreversibly converted to creatinine each day and excreted in the urine (Wyss and Kaddurah-Daouk, 2000). Consequently, creatine must be continually replaced (Lemme et al., 2007). The demand for creatine or its precursors may be supplied either directly from animal protein in the diet or by endogenous synthesis (Wyss and Kaddurah-Daouk, 2000). Creatine is produced naturally in the body from GAA, which in turn is synthesized from the amino acids arginine and glycine (Wyss and Kaddurah-Daouk, 2000). These amino acids are highly involved in muscle metabolism (Lemme et al., 2007). The marked growth-promoting effect of creatine may be most simply explained on an arginine-sparing basis (Dilger et al. 2013; DeGroot, 2014). Arginine and glycine are normally found in higher concentrations in feedstuffs such as animal by-products meal than in corn and soybean meal (Baker, 2009; Li et al., 2011). Poultry by-product meal (PBP) is still one of the most important sources of animal protein in animal feed (Bhargava and O’Neil, 1975; Meeker and Hamilton, 2006). Following the ban for the use of meat and bone meal in feed in 2001 (European Community, 2000), some European poultry producers observed a certain drop in performance (Costrel et al., 2009). This could have been partially caused by insufficient supply of creatine or its precursors Arg and Gly which are supposed to be present in animal derived proteins but absent in vegetable-based feed ingredients (Ringel et al., 2007). Approximately 90% of diets for poultry in the U.S. are comprised by corn and soybean meal (Baker, 2009). Vegetable protein sources have been reported (Gabor et al., 1984; Khan and Cowen, 1977) to have limited (< 0.01 mg/g) or no creatine content. However, animal by-products ingredient may have higher amounts of this nutrient depending on the type of manufacturing and quality (Harris et al., 1997; Dobenecker and Braun, 2015). In general, poultry by-products may contain low levels of creatine (Dobenecker and Braun, 2015), but 3.25 times more Gly and 32% more Arg than soybean meal (AminoDat 5.0). Consequently, broilers fed vegetable-based diets without animal by-products may face the possibility of lower supply of Arg and Gly for creatine endogenous production affecting muscle development. According to Petracci et al. (2015), novel pectoral myopathies, such as white striping (WS) and wooden breast (WB) are characterized by a reduced supply of oxygen and nutrients to muscle cell. In addition, this could also be associated with the inadequate elimination of metabolic intermediate products, leading to a membrane dysfunction and increased plasma concentration of enzymes such as creatine kinase (CK) and lactate dehydrogenase (LDH) (Mitchell, 1999; Sandercock and Mitchell, 2003; MacRae et al., 2006). Several management and nutritional solutions have been proposed to minimize and solve these myopathies and meat quality abnormalities. Among these strategies, the reduction of growth rate by using feed restriction programs, reduction of amino acids, lowering the energy value of the diets (Kuttappan et al., 2012a; Trocino et al., 2015; Radaelli et al., 2017), and higher dietary vitamin E and selenium (Guetchom et al., 2012; Kuttappan et al., 2012b) had been tested showing some benefits, improving meat quality and reducing occurrence of myopathies. However, the reduction of growth rate could depress productivity in broiler operations. Based on mice trials, it has been proposed that creatine may have a protective effect on certain neuromuscular (Tarnopolsky, 2007; Chung et al., 2007), and neuro-degenerative (Bender et al., 2006; Kolling and Wyse, 2010; Beal, 2011) diseases, and it also may reverse muscular dystrophy (Nabuurs et al., 2013). These results may be due to the capacity of creatine to prevent the inhibition of energy metabolism and lipid peroxidation. Therefore, our study evaluated a possible beneficial effect of GAA supplementation on pectoral myopathies without reducing the genetic potential for growth of broilers. Creatine production is a very important muscle metabolite and the introduction of an additive like GAA for creatine synthesis may influence blood markers related to broiler health and energy utilization and metabolism. Available published data regarding blood chemistry to assess metabolic status of heavy broilers fed PBP, and GAA, and compared with purely vegetable-based diets have not been reported so far. In the U.S., 47.5% of broilers are grown for more than 56 d (Agristats, 2016), therefore the present experiment focused on metabolites and breast meat quality parameters in heavy broilers of eight wk of age. Previous studies showed that GAA supplementation lowered breast meat pH, affecting the water holding capacity and improving the meat tenderness. Kuttappan et al. (2017) reported that WB and WS pectoral myopathies are highly correlated with meat quality parameters such us higher ultimate pH (>6.04), and drip loss. Considering that it has been reported that dietary GAA supplementation slightly affected ultimate pH in diets without fish meal (Michiels et al., 2012), there could be a possible beneficial effect on the occurrence of pectoral myopathies. Previous experiments regarding GAA supplementation did not evaluate the effects of dietary GAA inclusion on pectoral myopathies. Consequently, the objective of this study was to evaluate the effects of GAA supplementation on live performance, meat quality, metabolite serum concentrations, blood hematological profile and chemistry, and pectoral myopathies severity in heavy broilers fed corn-soybean meal based diets with or without PBP. MATERIALS AND METHODS Treatments and Birds Husbandry All the procedures involving the birds used in the present experiment were approved by the North Carolina State University Institutional Animal Care and Use Committee. Four dietary treatments from a 2 × 2 factorial arrangement with PBP and GAA as main effects were considered. Diets contained two levels of poultry by-products (0 vs. 5%), and two levels of GAA (0 and 0.06%). The study herein was conducted in an open-sided house with clear curtains and negative pressure ventilation. A total of 1,280 Ross-708-d-old male chicks were randomly placed in 64 floor pens (3.81 × 1.19 m) 16 pens per treatment and 20 chicks per pen (4.42 broilers/m2 at placement) for a final stocking density of 23.2 kg/m2 at 55d of age. Used litter was employed as bedding in the pens and broilers were exposed to continuous light on a 23L:1D (30 lux light intensity) program during the first seven d of age. Day length was then gradually reduced to 17L:7D (10 lux) up to 28 d of age. From 28 d until the end of the experiment, light program was maintained at 17L:7D with an intensity of 5 lux. At this age, natural daylight length lasted from 6:30 am to 5:30 pm, and supplemental light was offered from 5:30 pm to 9:00 pm. Brooding house temperature was set at 33.6°C at placement and gradually reduced until 20.6°C at 21 d and kept until d 56 to guarantee chicken environmental comfort. Diets Dietary treatments were obtained from two basal diets to ensure similar nutrient content among treatments in all experimental phases. Basal feeds consisted in corn-soybean meal based diets with or without the inclusion of PBP (0 vs. 5%). Subsequently, GAA was supplemented either 0 or 600 g of GAA per ton of feed (recommended dose, European Commission) in the form of CreAMINO® (Evonik Nutrition & Care Gmbh, Hanau, Germany) according to the corresponding treatment (Table 1). Diets that did not contain GAA had 600 g of sand per ton of feed. All dietary treatments were formulated to be isoenergetic, isonitrogenous, and mixed to represent typical U.S. broiler industry practices (AgriStats, 2016). Formulated digestible amino acid levels were based on AminoDat® 5.0 (2015) recommendations (Table 2). Macro ingredients (corn, soybean meal, and distilled dried grain with solubles) were analyzed for total amino acid and ME content prior to diet formulation. Digestible amino acid content was calculated from the total amino acid content obtained from lab analyses and using table values for digestibility coefficients (AminoDat 5.0, 2015). The ME values (kcal/kg) were obtained from an in vivo trial with roosters (Dr. Nick Dale, University of Georgia). Total and digestible amino acid content for PBP was obtained using the same procedures as described for the macro ingredients. Creatine-concentration in PBP diets were determined by AlzChem AG (Trosberg, Germany) according to the ion chromatography method (CRL Feed Additives, 2007). Starter, grower, finisher and withdrawal diets were fed from 0–14, 15–35, 36–42, and 43–56 d of age, respectively. Starter was fed in crumbles and all other diets in pellets. Table 1. Ingredient composition of starter, grower, finisher and withdrawal basal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00  1Coban® 90 (Monensin), Elanco Animal Health, Greenfield, IN, at 500 g/ton in the starter and grower diets. 2Trace minerals provided per kg of premix: manganese (Mn SO4), 60 g; zinc (ZnSO4), 60 g; iron (FeSO4), 40 g; copper (CuSO4), 5 g; iodine (Ca(IO3)2),1.25 g. 3Vitamins provided per kg of premix: vitamin A, 13,227,513 IU; vitamin D3, 3,968,253 IU; vitamin E, 66,137 IU; vitamin B12, 39.6 mg; riboflavin, 13,227 mg; niacin, 110,229 mg; d-pantothenic acid, 22,045 mg; menadione, 3,968 mg; folic acid, 2,204 mg; vitamin B6, 7,936 mg; thiamine, 3,968 mg; biotin, 253.5 mg. 4Quantum Blue 5G® at 0.176 lbs/ton (80 g/ton) to provide 500 FYT (AB Vista) delivering 0.13% of available P, 0.06% of calcium and 0.03% of sodium. 5CreAMINO®: Guanidinoacetic acid (GAA) with 96% of concentration, Lot numbers: 3/29/16, 9/26/16. View Large Table 1. Ingredient composition of starter, grower, finisher and withdrawal basal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Ingredient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    ————————————————————————%———————————————————–————  Corn  57.683  52.509  60.303  55.030  64.264  58.992  69.093  63.821  Soybean meal, 46%  25.899  33.973  23.114  31.316  19.219  27.421  15.047  23.249  Poultry By Products (PBP)  5.000  –  5.000  –  5.000  –  5.000  –  DDGs  5.000  5.000  5.000  5.048  5.000  5.000  5.000  5.000  Poultry fat  2.724  4.302  3.451  5.000  3.790  5.386  3.305  4.902  Limestone fine  1.212  1.366  1.013  1.167  0.942  1.096  0.955  1.109  Dicalcium phosphate, 18.5%  0.776  1.151  0.527  0.901  0.346  0.719  0.053  0.427  Salt (NaCl)  0.243  0.303  0.252  0.315  0.188  0.251  0.181  0.244  DL- Methionine, 99%  0.276  0.286  0.251  0.243  0.204  0.196  0.213  0.205  L-Lysine-HCl, 78.8%  0.270  0.218  0.236  0.170  0.183  0.118  0.264  0.198  Mineral premix2  0.200  0.200  0.200  0.200  0.200  0.200  0.200  0.200  Sodium bicarbonate  0.195  0.183  0.184  0.167  0.272  0.255  0.263  0.246  Choline chloride, 60%  0.180  0.180  0.180  0.180  0.180  0.180  0.180  0.180  Vitamin premix3  0.100  0.100  0.100  0.100  0.100  0.100  0.100  0.100  L-Threonine, 98%  0.088  0.081  0.070  0.045  0.044  0.020  0.077  0.053  Sand or GAA5  0.060  0.060  0.060  0.060  0.060  0.060  0.060  0.060  Coccidiostat1  0.050  0.050  0.050  0.050  –  –  –  –  L-Valine, 96.5%  0.036  0.031  0.001  0.001  0.001  –  –  –  Phytase4  0.008  0.008  0.008  0.008  0.008  0.008  0.008  0.008  Total  100.00  100.00  100.00  100.00  100.00  100.00  100.00  100.00  1Coban® 90 (Monensin), Elanco Animal Health, Greenfield, IN, at 500 g/ton in the starter and grower diets. 2Trace minerals provided per kg of premix: manganese (Mn SO4), 60 g; zinc (ZnSO4), 60 g; iron (FeSO4), 40 g; copper (CuSO4), 5 g; iodine (Ca(IO3)2),1.25 g. 3Vitamins provided per kg of premix: vitamin A, 13,227,513 IU; vitamin D3, 3,968,253 IU; vitamin E, 66,137 IU; vitamin B12, 39.6 mg; riboflavin, 13,227 mg; niacin, 110,229 mg; d-pantothenic acid, 22,045 mg; menadione, 3,968 mg; folic acid, 2,204 mg; vitamin B6, 7,936 mg; thiamine, 3,968 mg; biotin, 253.5 mg. 4Quantum Blue 5G® at 0.176 lbs/ton (80 g/ton) to provide 500 FYT (AB Vista) delivering 0.13% of available P, 0.06% of calcium and 0.03% of sodium. 5CreAMINO®: Guanidinoacetic acid (GAA) with 96% of concentration, Lot numbers: 3/29/16, 9/26/16. View Large Table 2. Calculated and analyzed nutrient content of basal starter, grower, finisher, and withdrawal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18  1Analyzed values are means of 2 samples. Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large Table 2. Calculated and analyzed nutrient content of basal starter, grower, finisher, and withdrawal diets for Ross-708 male broilers.   Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18    Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)  Nutrient  PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP  Calculated nutritive value   Metabolizable Energy, kcal/kg  3000  3000  3085  3085  3150  3150  3175  3175   Crude protein, %  22.11  22.11  20.95  20.95  19.31  19.31  17.80  17.80   Calcium, %  1.02  1.02  0.90  0.90  0.82  0.82  0.76  0.76   Total phosphorus, %  0.61  0.61  0.51  0.56  0.46  0.51  0.39  0.45   Nonphytate phosphorous, %  0.50  0.50  0.45  0.45  0.41  0.41  0.35  0.35   Total glycine, %  1.23  0.91  1.19  0.87  1.12  0.80  1.06  0.73   Digestible lysine, %  1.22  1.22  1.08  1.08  0.95  0.95  0.92  0.92   Digestible methionine, %  0.60  0.59  0.56  0.53  0.49  0.47  0.49  0.47   Digestible total sulfur amino acids, %  0.89  0.89  0.81  0.81  0.73  0.73  0.71  0.71   Digestible threonine, %  0.78  0.78  0.70  0.70  0.63  0.63  0.61  0.61   Digestible tryptophan, %  0.22  0.23  0.20  0.22  0.18  0.20  0.16  0.18   Digestible valine, %  0.94  0.94  0.83  0.86  0.80  0.83  0.74  0.77   Digestible arginine, %  1.32  1.35  1.19  1.25  1.08  1.14  0.97  1.03   Digestible isoleucine, %  0.89  0.93  0.73  0.78  0.67  0.72  0.60  0.65   Sodium, %  0.20  0.20  0.20  0.20  0.20  0.20  0.20  0.20   Potassium, %  0.84  0.94  0.77  0.89  0.70  0.82  0.63  0.75   Chloride, %  0.28  0.28  0.28  0.28  0.24  0.24  0.25  0.25   Dietary electrolyte balance, mEq/100 g  241  264  222  251  215  244  196  225  Analyzed nutritive value1   Crude protein, %  21.92  21.85  20.52  19.96  18.82  18.63  18.77  18.42   Total lysine, %  1.34  1.33  1.23  1.19  1.07  1.04  1.15  1.09   Total methionine, %  0.61  0.57  0.57  0.52  0.50  0.47  0.53  0.48   Total sulfur amino acids, %  0.93  0.91  0.88  0.84  0.78  0.77  0.83  0.78   Total threonine, %  0.88  0.88  0.81  0.81  0.72  0.70  0.77  0.74   Total glycine, %  1.08  0.90  1.03  0.84  0.95  0.77  0.96  0.77   Total valine, %  1.04  1.04  0.94  0.94  0.86  0.87  0.86  0.85   Total arginine, %  1.41  1.43  1.32  1.33  1.17  1.19  1.19  1.18  1Analyzed values are means of 2 samples. Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large For the pelleting process, a temperature between 82 and 85°C in the conditioner was used for 30 seconds. The steam pressure was 32 psi (221 kPa), and the pellet die was 11/64″ x 1″ 3/8″ (4.4 × 34.9 mm) for an L/D ratio of 8. The capacity of pelleting used was 2 to 5 ton/hour (2,000 to 5,000 kg per hour) to improve pellet quality. Representative samples of each final dietary treatment were collected after crumbling (starter) or pelleting process, to analyze creatine, and GAA content according to the IC method (CRL Feed Additives, 2007), and fully validated procedures (Dobenecker and Braun, 2015) by AlzChem AG, Trostberg, Germany (Table 3). For each one of the dietary phases 0.85, 2.90, and 2.48 kg of starter, grower and finisher, respectively, were offered for each bird alive during each phase. The withdrawal diet was offered ad libitum. Water was provided for ad libitum consumption. Feeders were shaken twice daily to stimulate uniform feed intake. Table 3. Calculated and analyzed creatine and GAA content of the dietary treatments for Ross-708 male broilers. Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  1Values are means of 2 samples. Report issued by the Alzchem AG according to the IC method (CRL Feed Additives, 2007) 2Calculated values (CreAMINO®, GAA min. 96%), Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large Table 3. Calculated and analyzed creatine and GAA content of the dietary treatments for Ross-708 male broilers. Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  Nutrient  Starter (0-14d)  Grower (15-35d)  Finisher (36-42d)  Withdrawal (43-56d)    PBP  No PBP  PBP  No PBP  PBP  No PBP  PBP  No PBP    None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  None  GAA  Calculated  GAA, mg/kg, as is  0  600  0  600  0  600  0  600  0  600  0  600  0  600  0  600  Analyzed1  Creatine, mg/kg, as is  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  <20  GAA, mg/kg, as is  30  518  <20  523  <20  630  <20  606  <20  563  <20  603  28  571  <20  565  CreAMINO®2, mg/kg, as is  28  540  <21  545  <21  656  <21  631  30  586  <21  628  30  595  <21  589  1Values are means of 2 samples. Report issued by the Alzchem AG according to the IC method (CRL Feed Additives, 2007) 2Calculated values (CreAMINO®, GAA min. 96%), Evonik Industries, Evonik Degussa GmbH, Hanau-Wolfgang, Germany. View Large Live Performance and Blood Collection Group BW was obtained at 0, 14, 35, 48, and 55 d of age. Feed intake, BW gain, and feed conversion ratio (FCR) were calculated at the end of each phase. Mortality was monitored and recorded twice daily. At 55 d, individual BW were obtained to calculate flock uniformity using the CV%. One broiler per pen was randomly selected to collect blood samples from non-fasting chickens. These samples were obtained from the brachial vein into plain, heparinized or EDTA containing tubes for hematologic profile with differential for white blood cells count (WBC), red blood cells count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), count of heterophils, thrombocytes, monocytes, lymphocytes, T-cell, B-cell. Differential blood count analysis (thrombocytes, heterophils, monocytes, lymphocytes, T-cells, and B-cells) was performed by LMU Ludwig-Maximilians University Laboratory in Munich, Germany, as described by Seliger et al. (2012) using a BD FACS Canto II instrument (Becton Dickinson, Heidelberg, Germany) for cell counting and BD FACS DIVA and FlowJo (Tree Star Inc., OR, USA) software for data analysis. For clinical hematology analysis and red blood cell count, samples were submitted to a commercial laboratory (Synlab Vet Laboratory, Cologne, Germany), to analyze erythrocytes, hemoglobin, hematocrit, MCV, MCH, and MCHC, using flow cytometry (FCM), and ADVIA 2120 analyzer from Siemens (Siemens, Deutschland GmbH, Hamburg, Germany). Serum enzyme activity of creatine kinase (CK), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT) and lactate dehydrogenase (LDH) were analyzed, as well as glucose, uric acid, minerals (phosphorus, calcium, sodium, potassium, and chloride), total protein, albumin, and cholesterol were also analyzed at North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA). Serum [creatine, creatinine, and GAA] and homocysteine in plasma were analyzed using standardized procedures (Baylor University, Houston, TX). Selection of Birds for Processing At 55 d, individual and group BW were obtained, and the average for each pen was calculated. Four broilers (including the selected for blood collection) per pen were selected with BW within two standard deviations above or under their corresponding average for each pen. Carcass and Cut up Yields At 56 d, feed was withdrawn for 12-h. Broilers were slaughtered at the NCSU pilot processing plant. Broilers were weighed, electrically stunned for 11 s, killed by exsanguination, and allowed to bleed for 90 s. Broilers were then scalded at 55°C for 90 s, picked for 30 s, and manually eviscerated. Carcasses were dressed by removing liver, gizzard, heart, oil gland, crop, proventriculus, lungs, and viscera. Carcasses were then air-chilled for six h, and manually deboned on stationary cones. Parts of the leg quarters, breast fillets (Pectoralis major), breast tenders (Pectoralis minor), wings, and rack with skin were obtained and weighed. The carcass yield was calculated for the chilled carcass as a percentage of the fasted live BW. Cut up yields were expressed as a percentage of the chilled absolute carcass BW. Meat Quality Evaluation To determine drip loss, the right Pectoralis major muscle was weighed six h post-mortem and immediately placed in a plastic bag, hung from a hook, and stored between 4–6°C for 24 h. After hanging, the sample was gently wiped with paper and weighed again. The difference in weight corresponded to the drip loss and was expressed as the percentage of the initial muscle weight. For cook loss, the left breast fillets (Pectoralis major) were weighed 24 h postmortem, placed on grilled-aluminum trays, and cooked in a forced air oven (SilverStar Southbend, Model SLES/10sc, gas type, NC, USA). Fillets were cooked to an internal temperature of 75°C (approximately 35 min), as measured by a Therma Plus thermocouple with a 10-cm needle temperature probe (ThermoWorks Model 221-071, UT, USA). The cooked fillets were cooled to room temperature, wiped gently with paper and re-weighed to determine cook yield as a percentage of the cooked weight relative to the raw weight. Shear force (kg) of cooked breast fillets samples was determined using a Warner-Bratzler shear device (Warner-Bratzler meat shear, Bodine Electric Company, Chicago, USA). Two samples per breast fillets (2 × 2 × 2 cm3) were sheared in a direction perpendicular to the muscle fibers. The maximum force measured when cutting the samples was expressed in kg force. Postmortem pH (t = 1, 6 and 24 h) was measured after skin was removed from Pectoralis major muscle samples using a portable pH meter (Oakton waterproof pH Tester 30). Color was measured on skinless Pectoralis major samples by the CIE L* (lightness), a* (redness), and b* (yellowness) system using a Minolta Chroma Meter CR-400 (Konica Minolta Sensing, Inc., Japan). A measuring area of 10 mm and illuminant D65 and 2° standard observer were used. The colorimeter was calibrated using a white tile (reference number 13033071; Y = 93.9, x = 0.3156, y = 0.3318). Pectoral Myopathies Skinless Pectoralis major muscle samples were visually evaluated to determine pectoral myopathies (spaghetti muscle, WB and WS) severity. The evaluation was performed by two experts in the field (College of Veterinary Medicine, North Carolina State University). Spaghetti muscle was recorded as presence or absence of this abnormality, whereas WS and WB were scored based on severity. The WB severity was based on a four-point scale as described by Tijare et al. (2016). Fillets samples were manually palpated and determined a score of 1 as a normal fillet with no WB signs, score 2 was considered a low severity, score 3 a medium, and score 4 as severe. The WS severity was scored with the scale used by Kuttappan et al. (2012c) which considered a three-point based scale of severity. Score 1 described a breast fillet with no white striations on the surface. Score 2 were the fillets with white striations less than 1 mm of thickness and easily observed in the surface, and score 3 was represented by the white striation more than 1 mm of thickness. In addition, the probability distribution for myopathies (WB and WS) scores was analyzed. Statistical Analysis Data were analyzed as completely randomized block design with a 2 × 2 factorial arrangement of treatments with the inclusion of PBP (0 vs. 5%), and GAA supplementation (0 vs. 0.06%) as main effects to have a total of four treatments. Each treatment respectively had 16 replicates distributed equally in four blocks (location of pens within the house) that were considered random effect. Data was analyzed in JMP 12 (SAS Institute. Inc., Cary, NC, 2016) using ANOVA in a mixed model. Differences between means were separated by the LS means method using Tukey's or t-student test at a level of significance of alpha = 0.05. Additional to this, for blood, carcass and cut up yields, meat quality and pectoral myopathies results, individual broiler's data from the same pen were nested inside every corresponding treatment and considered as random effect. Cutter was also included in the model as random effect for carcass and cut up yields. Data of scores probability distribution for WB and WS were analyzed using GLIMMIX (SAS Institute, 2008). RESULTS AND DISCUSSION Diets and Live Performance The analysis of GAA, and creatine are shown in Table 3. The GAA results showed slight differences on concentrations compared to the intended dose (600 mg/kg). The concentration of CreAMINO® in feed was calculated according to the standard concentration of GAA (CreAMINO®, GAA, 96% min, AlzChem AG, Trostberg, Germany) in the product. As expected, low or absence of creatine was obtained in all the dietary treatments (Table 3). Creatine analysis of PBP indicated a concentration of 156 mg/kg that did not affect final dietary concentration of creatine. Analyzed total amino acid and crude protein content were similar to formulated values and these are presented in Table 2. No interaction (P > 0.05) effects of treatments were observed on BW, BW gain, feed intake, and FCR in any of the phases evaluated (Table 4). Feed intake was not affected (P > 0.05) by PBP inclusion in the diet nor GAA addition throughout the whole experiment. At 0–14, 0–48 and 0–55 d, dietary inclusion of PBP decreased (P < 0.05) BW and BW gain. Broilers fed diets without PBP were heavier and gained more weight than broilers that were fed diets with PBP. The FCR was not affected (P > 0.05) by PBP or GAA at 0–14d (data not shown) and 0–35 d. However, at 0–48 and 0–55 d, FCR was improved (P < 0.01) up to two points by GAA supplementation regardless of the inclusion of PBP in the diet. At these ages, broilers fed diets with PBP had worse (P < 0.05) FCR than broilers fed diets without PBP. Diets without PBP and with GAA supplementation had slightly worse (P < 0.05) flock uniformity with higher CV% of individual BW. Total mortality was not affected (P > 0.05) by PBP inclusion or GAA supplementation throughout the experiment with no mortality in some pens. In general, overall average mortality rates per pen was close to 8% up to 55d including culled birds due to leg problems. Table 4. Effect of supplementation of guanidinoacetic acid (GAA) in corn-based diets with or without poultry by-products for Ross-708 male broilers on live performance up to 55 d of age.1 Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  1Values are means of 16 pens per treatment combination with 20 male broiler chickens. 2Flock uniformity at 55 d of age. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Table 4. Effect of supplementation of guanidinoacetic acid (GAA) in corn-based diets with or without poultry by-products for Ross-708 male broilers on live performance up to 55 d of age.1 Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  Diet type  Additive  BW  BW gain  Feed intake  FCR      0d  14d  35d  48d  55d  CV2  0-14d  0-35d  0-48d  0-55d  0-14d  0-35d  0-48d  0-55d  0-35d  0-48d  0-55d      ——————– (g) ——————–  —(%)—  ——————————— (g) ——————————–  —— (g:g) ——  No PBP    46.1  509a  2,673a  4,278a  5,058a  5.05a  463a  2,619  4,237a  5,012a  555  3,675  6,625  8,362  1.396  1.552b  1.650b  PBP    46.0  502b  2,650b  4,237b  5,014b  5.00b  456b  2,605  4,200b  4,966b  550  3,683  6,662  8,429  1.403  1.565a  1.673a  SEM    0.1  2  11  20  33  0.03  2  8  16  33  2  16.43  27  52.27  0.003  0.003  0.003    GAA  46.0  505  2,665  4,272  5,056  5.05a  459  2,617  4,235a  5,012a  551  3,684  6,634  8,373  1.396  1.551b  1.652b    None  46.0  506  2,659  4,243  5,016  5.00b  460  2,608  4,200b  4,966b  554  3,674  6,653  8,418  1.403  1.566a  1.671a    SEM  0.1  2  11  20  33  0.03  2  8  20  33  2  16.42  27  52.25  0.003  0.003  0.003  No PBP  GAA  46.1  506  2,679  4,287  5,076  5.07  460  2.627  4,248  5,031  551  3,686  6,605  8,335  1.394  1.548  1.643    None  46.1  511  2,668  4,270  5,041  5.03  465  2.612  4,227  4,994  559  3,664  6,646  8,389  1.398  1.557  1.658  PBP  GAA  46.0  504  2,651  4,257  5,036  5.04  458  2,606  4,222  4,993  552  3,683  6,663  8,411  1.398  1.554  1.661    None  46.0  500  2,650  4,217  4,992  4.96  454  2,605  4,173  4,939  549  3,683  6,661  8,447  1.408  1.576  1.685    SEM  0.1  3  13  23  36  0.03  3  10  23  35  3  21  35  61  0.005  0.005  0.005  CV%    0.61  2.42  1.57  1.48  1.66  1.68  2.65  1.37  1.48  1.67  2.19  1.99  1.81  2.03  1.46  1.25  1.29  Source of variation  ——————————————————— P – values ————————————————————  Diet type    0.152  0.020  0.036  0.012  0.043  0.017  0.0218  0.127  0.009  0.030  0.1745  0.682  0.234  0.127  0.183  0.013  <0.001  GAA    0.772  0.855  0.577  0.081  0.068  0.012  0.8382  0.386  0.021  0.034  0.4660  0.570  0.530  0.304  0.180  0.003  <0.001  Diet type*GAA    0.807  0.158  0.620  0.472  0.833  0.378  0.1453  0.464  0.351  0.687  0.0665  0.570  0.488  0.838  0.571  0.203  0.381  1Values are means of 16 pens per treatment combination with 20 male broiler chickens. 2Flock uniformity at 55 d of age. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Previous studies (Escalona and Pesti, 1987; Caires et al., 2010) concluded that PBP dietary inclusion did not affect live performance when including up to 5% in broiler diets. In the present experiment, the dietary inclusion of PBP resulted in reduced live performance. This result was unexpected, since the physical evaluation and laboratory analyses of nutrient content resulted in normal bromatological characteristics of this feed ingredient with no microbiological contamination. A possible over-estimation of digestible amino acid content in this feed ingredient may explain the observed response on live performance. Moreover, results reported by Michiels et al. (2012) suggested that no improvements on broiler live performance were observed when GAA supplemented diets were compared with a positive control diet containing fish meal. Conversely, Lemme et al (2011) concluded that dietary GAA improved live performance in diets containing fish meal. The present study confirmed that GAA at a level of 0.06% can effectively improve FCR and BW gain in diets containing animal meals, in this case PBP. Previous research validated the efficacy of dietary GAA inclusion on improving live performance in chickens that were fed diets containing only vegetable ingredients (Lemme et al., 2007; Ringel et al., 2007; Costrel et al., 2009; Michiels et al., 2012; DeGroot, 2014), as well as in arginine-deficient diets in young chicks due to the sparing effect of GAA on Arg (Savage and O΄|'Dell, 1960; Dilger et al., 2013; DeGroot, 2014; DeGroot et al., 2015). Therefore, dietary GAA supplementation improved live performance even in cases where Arg supply in feed was deficient. Results observed in the present study suggested that dietary GAA improved live performance regardless of the inclusion of PBP in the diet. Carcass Yield and Cut up Parts Dietary PBP and GAA showed interaction effects (P < 0.05) on Pectoralis major and breast meat yield. GAA supplementation improved Pectoralis major and breast meat yield only in diets containing PBP (Table 5). Broilers fed diets containing PBP and supplemented with GAA had higher (P < 0.05) yields compared to broilers fed non-supplemented diets with PBP. Carcass, wings, and leg quarters yields were not affected (P > 0.05) either by dietary PBP or GAA. Caires et al. (2010) observed no differences (P > 0.05) on carcass and breast meat yields when corn-soybean meal diets were compared to diets containing PBP (5%). Michiels et al. (2012) observed improvements (P < 0.05) on breast meat yield by up to 1.04-fold when GAA was added (0.06% and 1.2%) in corn-based diets. However, when treatments were compared with a positive control containing fish meal, this effect was not observed (P > 0.05). Interestingly, in the current trial the dietary GAA supplementation improved breast meat yield only in diets containing PBP. Table 5. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross-708 male broilers on carcass, breast meat, and cut up part yields at 56 d of age.1         Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602          Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602  1Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment and cutter (random effects) 2Relative to live weight of broilers selected for processing. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Table 5. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross-708 male broilers on carcass, breast meat, and cut up part yields at 56 d of age.1         Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602          Cut – up parts relative to carcass weight  Diet  Additive  Live  Carcass  Wings  Leg  Pectoralis  Pectoralis  Breast  Rack  type    weight  yield2    Quarters  major  minor  meat        — (g) —  ——————————————————– % ———————————————————  No PBP    4,975  78.72  9.41  29.83  32.14  6.29  38.39  22.41  PBP    4,948  78.63  9.26  30.21  32.04  6.38  38.39  22.30  SEM    28  0.14  0.05  0.32  0.21  0.06  0.21  0.11    GAA  5,006a  78.62  9.34  29.92  32.13  6.36  38.44  22.43    None  4,917b  78.72  9.33  30.11  32.05  6.32  38.34  22.28    SEM  28  0.14  0.05  0.32  0.21  0.06  0.21  0.11  No PBP  GAA  5,016  78.53  9.39  29.92  31.92a,b  6.29  38.20a,b  22.32    None  4,934  78.72  9.43  29.73  32.36a  6.30  38.59a  22.27  PBP  GAA  4,995  78.73  9.30  29.92  32.35a  6.43  38.68a  22.55    None  4,900  78.72  9.22  30.49  31.74b  6.34  38.09b  22.28    SEM  36  0.18  0.08  0.35  0.26  0.07  0.27  0.18  CV%    5.66  5.58  6.42  4.39  5.45  7.21  4.88  6.28  Source of variation  ——————————————– P – values ————————————————  Diet type    0.394  0.545  0.085  0.069  0.623  0.186  0.968  0.574  GAA    0.009  0.512  0.888  0.366  0.687  0.550  0.664  0.437  Diet type*GAA    0.846  0.553  0.476  0.070  0.013  0.417  0.034  0.602  1Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment and cutter (random effects) 2Relative to live weight of broilers selected for processing. a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. View Large Meat Quality Generally, meat quality was not affected either (P > 0.05) by dietary PBP or GAA, except for an interaction effect (P < 0.05) observed on cook loss, and an effect on the b* (yellowness) value due to PBP inclusion in the diet. Broilers fed diets with PBP and not supplemented with GAA had the lowest (P < 0.05) cook loss (Table 6). Broilers fed diets with PBP had higher (P < 0.05) b* value than the ones fed diets without PBP (10.06 vs. 9.27). Table 6. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products on meat quality parameters at 56 d of age for Ross-708 male broilers.* Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  *Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment (random effects) a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. 1Warner-Bratzler; Color: L* = lightness, a* = redness, b* = yellowness 2(3 = severe; 2 = medium; 1 = low) 3(4 = severe; 3 = medium; 2 = low; 1 = normal) View Large Table 6. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products on meat quality parameters at 56 d of age for Ross-708 male broilers.* Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  Diet      Cook  Drip  Shear    White  Wooden  type  Additive  Post-mortem pH  loss  loss  force1  Colorimeter parameters  striping2  breast3      6 h  24 h        L*  a*  b*              —–(%)—–  —(kg)—        –(1-3)–  –(1-4)–  No PBP    6.01  5.99  19.30  1.08  4.40  55.59  6.11  9.27b  1.86  2.60  PBP    6.00  6.01  17.82  1.14  4.38  55.37  6.10  10.06a  1.98  2.49  SEM    0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07    GAA  6.02  6.00  18.76  1.19  4.42  55.58  6.18  9.66  1.94  2.49    None  5.99  6.00  18.36  1.04  4.37  55.38  6.04  9.68  1.90  2.59    SEM  0.02  0.02  0.42  0.19  0.12  0.50  0.43  0.33  0.13  0.07  No PBP  GAA  6.02  5.98  18.65a,b  1.17  4.36  55.70  6.20  9.13  1.90  2.47    None  6.00  6.00  19.95a  1.00  4.44  55.49  6.02  9.41  1.81  2.72  PBP  GAA  6.02  6.03  18.87a,b  1.20  4.47  55.46  6.16  10.19  1.97  2.51    None  5.98  5.99  16.76b  1.08  4.30  55.27  6.05  9.94  1.99  2.47    SEM  0.03  0.03  0.62  0.21  0.16  0.60  0.48  0.42  0.15  0.09  Source of variation  ————————————————— P – values ———————————————–  Diet type    0.688  0.308  0.027  0.674  0.951  0.628  0.978  0.040  0.240  0.257  GAA    0.275  0.773  0.540  0.299  0.757  0.680  0.650  0.963  0.694  0.262  Diet type*GAA    0.682  0.169  0.012  0.890  0.435  0.979  0.920  0.485  0.598  0.117  *Values are means of 16 pens per treatment combination with 4 male broiler chickens per pen selected for processing. Pen nested within each treatment (random effects) a,bMeans in a column not sharing a common superscript are significantly different (P ≤ 0.05) by Student's t or Tukey's test. 1Warner-Bratzler; Color: L* = lightness, a* = redness, b* = yellowness 2(3 = severe; 2 = medium; 1 = low) 3(4 = severe; 3 = medium; 2 = low; 1 = normal) View Large Fletcher et al. (2000) concluded that the ultimate pH of normal chicken breast meat ranges between 5.7 and 5.96. No differences (P > 0.05) due to treatments were observed in the present experiment and results indicated that ultimate pH ranged from 5.99 to 6.03. Michiels et al. (2012) also did not observed effects of GAA supplementation on postmortem pH, shear force, and a* value in diets containing fish meal. Generally, our measurements of shear force (Warner-Bratzler) ranged from 42.2 to 43.8 N (4.30 to 4.47 kg), which are values that can be considered as “moderately to slightly tender” category based on Lyon and Lyon (1991) sensory ranges. In addition, Poole et al. (1999) found that broilers around 56 d of age should have an average value of shear force (Warner-Bratzler) of about 4.64 kg ± 0.18 kg. They suggested that breast fillets in the scale from 3.46 to 6.41 kg are considered “moderate tender” which are similar values to the ones observed in the current experiment. Fletcher (2002) reported that differences in tenderness depend on age of the chickens and can be due to the fact that heavier broilers are more mature at the time of harvest and have more cross-linking of collagen. Therefore, results of our experiment suggested that the values of shear force obtained were related to age of the chickens at processing. Results obtained in b* value of the study herein could be explained by the level of inclusion of corn in the diet, and the theoretically carotenoid content in PBP (Sajilata et al., 2008). All experimental diets containing PBP had higher inclusion of corn, compared to diets that did not contain PBP throughout the whole experiment. Consequently, they contained a greater content of carotenoids in the feed that might have been deposited later in breast muscle. Pectoral Myopathies The occurrence (presence or absence) of “spaghetti muscle” was evaluated, but not enough breast fillets were affected with this myopathy to have enough replicates for statistical analysis. The WS and WB overall average scores were not affected (P > 0.05) either by dietary PBP inclusion or GAA supplementation (Table 6). However, an interaction effect (P < 0.05) was detected on the probability score distribution (Figure 1) for WB score 2 (low severity). In diets without PBP, the supplementation with GAA increased the probability of having WB score 2 up to two fold compared to samples from broilers fed non supplemented diets. This response is explained mainly by the reduction of score 3 due to GAA supplementation in diets without PBP. Additionally, in diets containing PBP the probability for WB score 2 was not different between treatments. No effects (P > 0.05) due to PBP inclusion or GAA supplementation were observed on the probability distribution for WS myopathy. Figure 1. View largeDownload slide Interaction effect of dietary PBP and GAA supplementation on probability distribution (0.00 – 1.00) for each wooden breast severity score in Ross-708 male broilers at 56 d of age. Means not sharing a common superscript (a-b) are significantly different (P < 0.05) by Tukey's test. Each value represents the probability (0.0 – 1.0) of developing each severity score according to main factors or factorial arrangement of treatments, n = 52 within 16 replicates per treatment combination. Scores are based on a 4-point scale (4 = severe, 3 = medium, 2 = low, 1 = normal). Figure 1. View largeDownload slide Interaction effect of dietary PBP and GAA supplementation on probability distribution (0.00 – 1.00) for each wooden breast severity score in Ross-708 male broilers at 56 d of age. Means not sharing a common superscript (a-b) are significantly different (P < 0.05) by Tukey's test. Each value represents the probability (0.0 – 1.0) of developing each severity score according to main factors or factorial arrangement of treatments, n = 52 within 16 replicates per treatment combination. Scores are based on a 4-point scale (4 = severe, 3 = medium, 2 = low, 1 = normal). According to Kuttappan et al. (2017), pH of breast meat affected with either WS, WB or both, presented higher (>6.04), ultimate pH values compared to samples without signs of these abnormalities. Khan (1974) concluded that meat pH is determined by muscle glycogen content and its degradation rate. Mudalal et al. (2014) suggested that GAA supplementation could avoid glycogen depletion and improve meat quality. Recently, Majdeddin et al. (2017) showed that higher phosphocreatine, creatine and glycogen concentrations in breast muscle of broilers were observed with supplementation of GAA (0.06 and 0.12%). Consequently, our results on WB may be a response driven by phosphocreatine and creatine more than by glycogen since no changes on ultimate pH were observed. Previous studies (Ringel et al., 2007; Michiels et al., 2012; DeGroot, 2014) showed that GAA supplementation increased the concentration of creatine in breast muscle. Intramuscular phospho-creatine can attract water into the muscle cell and increase the cell volume (Hultman et al., 1996). Haussinger (1996) found that a super-hydrated muscle may trigger protein synthesis, minimize protein breakdown, and increase glycogen synthesis. Our results may be explained by the protective effects of creatine found in muscle energy metabolism as reported in previous studies in humans (Balsom et al., 1994), rats (Kolling and Wyse, 2010; Kolling et al., 2013; Nabuurs et al., 2013) with muscle dystrophies (Pearlman and Fielding, 2006; Chung et al., 2007; Tarnopolsky, 2007). Nabuurs et al. (2013) concluded that muscular dystrophy could be reversed in rats by dietary supplementation of creatine. Kolling et al. (2013) suggested that the effect of creatine and its interaction with homocysteine altered glucose oxidation and protected muscle from energy imbalances in rats. It has been demonstrated that alterations in energy metabolism seem to be implicated in the pathogenesis of several muscle and neurological complications, metabolic disorders, aging and neuromuscular diseases including WB in broilers (Abasht et al., 2016). Therefore, the supplementation with GAA as a precursor of creatine could reduce the WB severity by supplying enough creatine avoiding glycogen depletion and consequently preventing muscle damage. Blood Analysis Blood results (Table 7) showed interaction effects (P < 0.05) of treatments only on GAA (Table 8) and MCV (Table 9) serum concentration. Broilers fed diets containing PBP without GAA supplementation had the lowest (P < 0.05) MCV serum concentration. Serum GAA concentration of broilers fed diets with PBP and GAA addition were 12.4 times higher (P < 0.05) than chickens fed non-supplemented diets. Broilers fed diets without PBP and GAA dietary supplementation increased the blood GAA up to 10 times compared to chickens fed non-supplemented diets. These results confirmed effective GAA uptake and metabolism independently of PBP inclusion. Hematological results (Table 9) of blood cells count were not affected (P > 0.05) by PBP inclusion nor GAA supplementation. Serum cholesterol concentration (Table 7) in broilers fed diets with PBP (140.34 mg/dl) was higher (P = 0.05) than chickens fed diets without PBP (133.15 mg/dl). On the other hand, other blood (Tables 7, 8, and 9) parameters (albumin, protein, glucose, uric acid, HBE, MCH, homocysteine), minerals (P, Ca, Na, K, Cl), and hepatic enzymes (ALT, AST, GGT, LD, CK) were not affected (P > 0.05) either by PBP inclusion in the diet or GAA supplementation. However, trends presented in Tables 7, 8 and 9 were observed on serum concentration of glucose (P = 0.06), chloride (P = 0.079), erythrocytes (P = 0.058), and hematocrit (P = 0.063) due to dietary PBP inclusion, and a trend in GGT (P = 0.069) attributed to GAA supplementation was found. Creatine serum concentration was higher (P < 0.01) in broilers fed supplemented diets than chickens fed non-supplemented diets (65.87 vs. 41.46 μM) due to dietary GAA. Moreover, broilers fed diets containing PBP had higher (P < 0.01) serum creatinine concentration compared to broilers fed diets without PBP (0.79 vs. 0.36 μg/ml). Table 7. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum metabolites at 55 d.1 Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Analysis were performed at North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA). View Large Table 7. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum metabolites at 55 d.1 Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  Diet type  Additive  Cholesterol  Glucose  Uric Acid  Albumin  Protein  Minerals                P  Ca  Na  K  Cl      ——(mg/dL)—–  ——(g/dL)——  ——(mg/dL)——  —–(mmol/L)—–  No PBP    133.15b  273.09  4.92  1.39  3.10  7.16  11.16  156.86  5.63  111.04  PBP    140.34a  263.27  4.83  1.45  3.09  7.04  11.09  155.85  5.87  109.60  SEM    2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70    GAA  136.05  267.78  5.05  1.43  3.13  7.14  11.15  156.01  5.69  110.07    None  137.44  268.59  4.70  1.41  3.05  7.07  11.10  156.69  5.82  110.57    SEM  2.33  3.35  0.28  0.04  0.07  0.08  0.14  0.60  0.12  0.70  No PBP  GAA  131.67  274.37  4.95  1.40  3.14  7.26  11.22  156.47  5.49  110.64    None  134.63  271.81  4.90  1.39  3.05  7.07  11.10  157.25  5.78  111.44  PBP  GAA  140.44  261.19  5.16  1.47  3.13  7.01  11.08  155.56  5.89  109.50    None  140.25  265.36  4.50  1.43  3.04  7.07  11.10  156.13  5.86  109.70    SEM  3.44  4.91  0.39  0.05  0.08  0.12  0.18  0.84  0.17  0.90  CV%    10.41  7.40  29.96  10.36  8.99  6.46  4.90  2.12  11.94  2.86  Source of variation  ——————————————— P – values ———————————————  Diet type    0.050  0.060  0.795  0.138  0.891  0.323  0.603  0.237  0.185  0.079  GAA    0.702  0.874  0.352  0.571  0.252  0.591  0.729  0.426  0.494  0.535  Diet type*GAA    0.664  0.512  0.423  0.671  0.999  0.298  0.622  0.901  0.370  0.710  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Analysis were performed at North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA). View Large Table 8. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum liver and muscle enzymes and metabolites at 55 d.1 Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a–cMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (gamma-glutamyl transpeptidase), LDH (lactic acid dehydrogenase), CK (creatine kinase). Analysis were performed by North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA), and Baylor University, Houston, TX. View Large Table 8. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on serum liver and muscle enzymes and metabolites at 55 d.1 Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  Diet type  Additive  ALT  AST  GGT  LDH  CK  Creatinine  Homocysteine  Creatine  GAA      —————————(IU/L) ————————  –(μg/ml) –  ——————(μM) —————–  No PBP    6.53  651.96  13.57  2,562  64,885  0.36b  15.14  50.12  3.34b  PBP    5.88  570.16  14.54  2,263  55,878  0.79a  16.59  57.21  4.53a  SEM    0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33    GAA  6.43  639.38  13.19  2,432  63,536  0.65  16.65  65.87a  7.09a    None  5.98  582.73  14.92  2,393  57,227  0.50  15.07  41.46b  0.77b    SEM  0.49  50.23  0.52  456  10,360  0.05  1.01  5.06  0.33  No PBP  GAA  6.92  688.82  12.14  2,599  70,443  0.36  16.14  58.25  6.01b    None  6.14  615.09  15.00  2,525  59,326  0.37  14.13  41.98  0.66c  PBP  GAA  5.94  589.94  14.25  2,265  56.628  0.95  17.17  73.48  8.18a    None  5.81  550.38  14.84  2,261  55,127  0.64  16.01  40.94  0.88c    SEM  0.57  66.05  0.83  554  11,976  0.08  1.46  6.65  0.45  CV%    25.89  40.13  25.93  72.50  55.62  60.35  37.54  45.05  43.19  Source of variation  ————————— P – values ———————–  Diet type    0.115  0.191  0.300  0.505  0.294  <0.001  0.337  0.250  0.007  GAA    0.275  0.363  0.069  0.931  0.461  0.097  0.297  <0.001  <0.001  Diet type*GAA    0.428  0.783  0.227  0.938  0.574  0.072  0.779  0.189  0.025  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a–cMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: ALT (alanine aminotransferase), AST (aspartate aminotransferase), GGT (gamma-glutamyl transpeptidase), LDH (lactic acid dehydrogenase), CK (creatine kinase). Analysis were performed by North Carolina State University, College of Veterinary Medicine Diagnostic Laboratory (Raleigh, NC, USA), and Baylor University, Houston, TX. View Large Table 9. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on hematological parameters at 55 d.1 Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration). 2Flow cytometry method. 3Results calculated. LMU Ludwig-Maximilians University Laboratory in Munich, Germany, and Synlab Vet Laboratory, Cologne, Germany. View Large Table 9. Effect of supplementation of guanidinoacetic acid (GAA) in diets with or without poultry by-products for Ross 708 broilers on hematological parameters at 55 d.1 Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  Diet type  Additive  Erythrocytes2  Hemoglobin2  Hematocrit3  MCV2  HBE (MCH)3  MCHC3  Thrombocytes  Heterophils  Monocytes  Lymphocytes  T-cells  B-cells      (T/l)  (g/l)  (%)  Fl  pg  g/dl  ———————– (cells/μl of blood) ——————————  No PBP    2.38  98.27  0.28  105.67  41.58  35.88  40,861  11,841  949  16,424  14,198  2,193  PBP    2.50  102.22  0.29  104.84  40.77  35.04  38,988  10,552  724  15,593  13,363  2,215  SEM    0.04  4.71  0.01  0.49  1.43  1.28  6,517  1,799  222  1,672  1,468  304    GAA  2.43  100.11  0.28  105.36  41.16  35.50  33,980  10,550  624  15,067  12,928  2,083    None  2.44  100.37  0.28  105.15  41.19  35.43  45,869  11,843  1,050  16,950  14,633  2,325    SEM  0.04  4.71  0.01  0.48  1.43  1.28  6,242  1,833  231  1,704  1,495  310  No PBP  GAA  2.39  96.65  0.27  104.79a,b  40.56  35.38  36,914  11,321  583  16,455  14,045  2,333    None  2.36  99.89  0.28  106.54a  42.59  36.39  44,807  12,360  1,314  16,394  14,351  2,054  PBP  GAA  2.47  103.58  0.29  105.92a,b  41.57  35.62  31,047  9,779  664  13,680  11,811  1,834    None  2.52  100.86  0.29  103.75b  39.79  34.47  46,930  11,326  785  17,507  14,914  2,596    SEM  0.06  5.63  0.01  0.82  1.77  1.59  9,099  2,449  307  2,272  1,975  405  Source of variation  —————————————————P – values ———————————————————–  Diet type    0.058  0.368  0.063  0.374  0.586  0.531  0.858  0.583  0.438  0.702  0.654  0.954  GAA    0.894  0.953  0.823  0.820  0.981  0.959  0.264  0.598  0.181  0.409  0.384  0.539  Diet type*GAA    0.497  0.500  0.969  0.043  0.186  0.428  0.691  0.912  0.332  0.393  0.473  0.192  1Values are means of 16 pens per treatment combination with 1 male broiler chicken per pen selected for blood collection a,bMeans followed by different superscripts within a column are significantly different (P < 0.05). Abbreviations: MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration). 2Flow cytometry method. 3Results calculated. LMU Ludwig-Maximilians University Laboratory in Munich, Germany, and Synlab Vet Laboratory, Cologne, Germany. View Large Zhang et al. (2017) reported that certain values in hematology may be affected by dietary GAA depending on dose and diet composition. DeGroot (2014) found no effects on blood cell count (leukocytes, heterophils, lymphocytes, monocytes, eosinophils, basophils), and only changes on differential cell proportions in heterophils and lymphocytes due to GAA supplementation in broilers fed both arginine-deficient and arginine-adequate diets. Previous trials (Tossenberger et al., 2016) showed that serum protein, albumin, cholesterol, glucose, urea, uric acid and enzymes ALT, AST, and GGT remained essentially unchanged (P > 0.05) even when dietary GAA inclusion was up to 0.6%. Likewise, Michiels et al. (2012) found no differences on serum glucose and uric acid in broilers fed corn-based diets without fish-meal and raised up to 26 d. According to DeGroot (2014), no differences (P > 0.05) attributed to GAA supplementation were detected on AST, CK and mineral (Ca, P) serum concentrations. In the experiment here in, the supplementation with GAA only increased GAA and creatine serum concentrations. Consequently, our results are in agreement with previous reports. Abasht et al. (2016) reported higher levels of gamma-glutamyl amino acid catabolite 5-oxoproline in tissue of samples affected by WB up to 1.57-fold as compared to unaffected samples. This suggests the import of extracellular glutathione that together with other biomarkers indicated muscle degradation and oxidative stress. Considering our findings, we may hypothesize that GAA reduced WB severity and the lower GGT serum blood level is related to this effect, despite of no effects on CK. Similar to our findings, DeGroot (2014) found that dietary GAA increased (P < 0.01) creatine and GAA serum concentration. Petracci et al. (2015) discussed that the presence of pectoral myopathies may be related by an increased concentration of CK and LDH. Although, reductions in WB severity were observed in the present experiment, no effects on serum concentration of these enzymes were detected due to GAA supplementation or PBP inclusion. This study showed generally no difference in endpoints determined in blood other than GAA and creatine which is expected. Furthermore, our findings provide evidence that dietary GAA supplementation improved broiler live performance up to 55 d, independently of PBP inclusion in the diets. 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Poultry ScienceOxford University Press

Published: Apr 13, 2018

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