
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00684-9
10.1016/j.psj.2024.104105
104105
METABOLISM AND NUTRITION
Effect of arginine, glycine + serine concentrations, and guanidinoacetic acid supplementation in vegetable-based diets for chickens
de Souza Cleison *
de Souza Cleverson *
Campos Felipe P. *
Savaris Vaneila. D.L. *
Wachholz Lucas *
Kaufmann Cristine *
Broch Jomara *
Comin Gabriel N. *
Calderano Arele A. †
Tesser Guilherme L.S. ‡
Starkey Jessica D. ‡
Eyng Cinthia *
Nunes Ricardo V. nunesrv@hotmail.com
*1
⁎ Department of Animal Science, Western Paraná State University, Marechal Cândido Rondon, PR 85960-000, Brazil
† Department of Animal Science, Federal University of Viçosa, Viçosa, MG 36570-900, Brazil
‡ Department of Poultry Science, Auburn University, Auburn, AL 36849, US
1 Corresponding author: nunesrv@hotmail.com
16 7 2024
10 2024
16 7 2024
103 10 1041051 3 2024
12 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The study investigated guanidinoacetic acid (GAA) supplementation with varying dietary digestible arginine (Arg) and glycine+serine (Gly+Ser) concentrations in the starter phase, exploring respective carry-over effects on growth performance, blood chemistry, incidence of pectoral myopathies and proximate composition in broilers. A total of 2,800 one-day-old male broiler chicks were distributed in a central composite design with 2 factors and double experimental mesh, represented by supplementation or omission of 0.6 g per kg of GAA, with a central point represented by 107% of Arg and 147% of Gly+Ser, 4 factorial points (combinations of Arg/Gly+Ser concentrations: 96.4/132.5%; 117.6/132.5%; 96.4/161.5%, and 117.6/132.5%), and 4 axial points (combinations of axial points estimated for Arg and Gly+Ser, with the central points of 92/147%; 122/147%; 107/126.5, and 107/167.5%), totaling 18 treatments, 4 repetitions to factorial and axial points, 24 replicates to the central point, and 25 birds per pen. Feed conversion ratio (FCR) from d 1 to 10 had a linear response (P = 0.009) for the decreasing Arg content and a quadratic response (P = 0.047) for Gly+Ser concentrations. Broilers supplemented GAA had lower FCR compared with nonsupplemented groups from d 1 to 10 (P = 0.048) and d 1 to 42 (P = 0.026). Aspartate aminotransferase (AST) exhibited increasing and decreasing linear effects as a function of Arg (P = 0.008) and Gly+Ser (P = 0.020) concentrations, respectively. Guanidinoacetic acid decreased serum AST (P = 0.028). Guanidinoacetic acid reduced moderate + severe (P = 0.039) and mild (P = 0.015) Wooden Breast scores. The occurrence of normal White Striping increased (P = 0.002), while severe score was reduced (P = 0.029) with GAA supplementation. In conclusion, increased digestible Arg:Lys and 14% and 6% above the recommendations (107% and 147%), respectively, provided improved FCR during the starter phase. Dietary GAA supplementation (0.6 g per kg) improved FCR, reduced severity of breast myopathies and appears to have reduced muscle damage in broilers fed plant-based diets.

Key words

amino acid
creatine
muscular dystrophy
white striping
wooden breast
==== Body
pmcINTRODUCTION

The poultry industry faces several productive challenges. Among these, the incidence of myopathies is particularly noteworthy due to their substantial impact, causing large economic losses worldwide (Zanetti et al., 2018). Myopathies can lead to alterations in the meat composition, reducing the protein content, while increasing fat and connective tissue, impairing the general consumers acceptance (Mudalal et al., 2014; Petracci et al., 2014; Velleman e Clark, 2015; Lucca et al., 2019).

Breasts affected by Wooden Breast (WB) have pale and protrude hardened areas on the surface, sometimes covered by slightly cloudy viscous fluid and multifocal petechiae (Mudalal et al. 2015). White Striping (WS) is the occurrence of visible white lines that run parallel to the breast muscle with varying proportion and thickness (Kuttappan et al. 2012). Studies have shown that breasts affected by WB or WS are characterized by a state of severe hypoxia (Mutryn et al., 2015; Boerboom et al., 2018), increased free radical composition and reduced muscle glycogen and glycolytic metabolites (Abasht et al., 2016). In addition, Arg conversion into citrulline (Arg-via oxide nitric) is one of the most impaired metabolic pathways in breasts affected by WS (Boerboom et al., 2018).

The creatine (CR) phosphocreatine system, serves as an important regulator and backup of ATP concentrations in tissues with high energy demand, such as the heart, skeletal muscle, and brain (Wallimann et al., 2011; Curt et al., 2015). Creatine can be endogenously synthesized from the amino acids Arg, Gly, and Met (Michiels et al., 2012; Curt et al., 2015). Additionally, CRE can be obtained exogenously from the food, with animal protein ingredients containing a significant amount of CRE, while plant-based foods are typically poor sources of this nutrient, increasing the exigence of Arg, Gly, and Met (Mousavi et al., 2013; Curt et al., 2015). According to Khajali et al. (2020), producing 1 g of GAA requires 1.49 g of Arg and 0.64 g of Gly.

Guanidinoacetic acid (GAA) is a naturally occurring amino acid acknowledged for its key involvement in the synthesis of CRE. Dietary GAA can act as a precursor of CRE, increasing its concentration and the phosphocreatine-to-ATP ratio in the muscle, improving muscle performance and yield, as well as actively spares Arg and Gly (Michiels et al., 2012; Córdova-Noboa et al., 2018; DeGroot et al., 2018; Khajali et al., 2020; Khajali et al., 2020; Sharma et al., 2022).

Research indicates that GAA has been associated with a reduced occurrence of breast myopathies, along with enhancements in live performance and breast meat yield (Córdova-Noboa et al., 2018; Vargas, 2019). Given this scenario, we hypothesized that the concentrations of digestible Arg and Gly in the starter diet phase in conjunction with GAA supplementation may influence the performance of broilers and the occurrence of myopathies. In addition, we hypothesized that dietary inclusion of GAA could have a Arg and Gly-sparing effect.

Therefore, this study was conducted to investigate the effect of GAA supplementation at varying dietary digestible Arg and Gly+Ser concentrations in the starter phase on overall performance traits, incidence of pectoral myopathies, and proximate composition in broilers fed plant-based diets.

MATHERIALS AND METHODS

Care and Use of Animals

This experiment was conducted at the Poultry Research Center of Western Paraná State University - Unioeste, Marechal Cândido Rondon, Paraná, Brazil. All the experimental procedures were approved by the Animal Use Ethics Committee of Unioeste under the protocol number 23/18. Euthanasia procedures were performed in accordance with the Normative Resolution n. 37 of February 2018, which establishes the euthanasia practice guidelines of the National Council for Animal Experimentation Control.

Experimental Treatments and Facilities

A total of 2,800 one-day-old male Ross 708 broiler chicks with an initial weight of 44.67 g (± 1.6 g) were used. Birds were vaccinated in the hatchery against Marek's disease, infectious bursal disease, fowlpox, and infectious bronchitis.

The broilers were distributed in a central composite design rotated with 2 factors, and double experimental mesh. Each experimental mesh, represented by on top inclusion of 0.6 g per kg of GAA or absence (Creamino, GAA 96% (min), Alzchem Ag, Trostberg, Germany) in the diet, presented 1 central point, 4 factorial and axial points. The central point of each experimental mesh was represented by 107% digestible Arg and 147% Gly+Ser (recommendations). The 4 factorial points were the combination of 2 digestible Arg with 2 Gly+Ser concentrations (96.4/132.5%; 117.6/132.5%; 96.4/161.5% and 117.6/132.5%).

The four axial points were calculated by interpolation, using the formula:α=2n/4=22/4=2=1,4142

Where: n = number of factorials.

The four axial points were the combination of 2 digestible Arg and 2 Gly+Ser estimated points with the central points (92/147%; 122/147%; 107/126.5 and 107/167.5%), totaling 18 treatments. Each factorial and axial point had 4 repetitions and the 2 central points had 24 repetitions each, all with 25 birds per pen. The percentage of digestible amino acids (Arg and Gly+Ser) were compared to the amount of digestible lysine (13.07 g per kg).

The isocaloric experimental diets were formulated based on corn and soybean meal according to the composition of feed ingredients and nutritional requirements proposed by Rostagno et al. (2017), for broiler chickens from 1 to 10 d of age, except for the crude protein and digestible amino acids (Arg and Gly+Ser (Table 1). On d 10, experimental diets were removed, and birds were fed a commercial diet until 42 d of age, while GAA supplementation was maintained. All experimental diets were fed in mash form.Table 1 Composition and nutrient specification (g per kg as-fed) of experimental diets for broiler chickens in the starter phase (d 1–10).

Table 1Ingredients	Arg1	96.4/132.5	117.6/132.5	96.4/161.5	117.6/161.5	92/147	122/147	107/126.5	107/167.5	107/147	
Gly	
Corn, 7.88%	607.0	606.5	605.1	605.1	606.5	606.5	606.5	605.1	605.1	
Soybean meal, 45%	314.1	314.1	314.1	314.1	314.1	314.1	314.1	314.1	314.1	
Soybean oil	12.59	13.09	14.49	14.49	13.09	13.09	13.09	14.49	14.49	
Dicalcium phosphate	19.32	19.32	19.32	19.32	19.32	19.32	19.32	19.32	19.32	
Limestone	10.72	10.72	10.72	10.72	10.72	10.72	10.72	10.72	10.72	
NaCl	4.00	4.00	4.00	4.00	4.00	4.00	4.00	4.00	4.00	
Glutamic acid	10.0	6.02	6.99	3.71	10.00	5.34	7.67	5.18	5.02	
L-Lys HCL, 78.4%	4.97	4.97	4.97	4.97	4.97	4.97	4.97	4.97	4.97	
DL-Met	4.45	4.45	4.45	4.45	4.45	4.45	4.45	4.45	4.45	
L-Thr	2.27	2.27	2.27	2.27	2.27	2.27	2.27	2.27	2.27	
L-Val	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	
L-Ile	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	
L-Gly	1.22	1.22	5.06	5.06	3.13	3.13	0.42	5.85	3.14	
L-Arg	0.62	3.60	0.63	3.60	0.00	4.22	2.11	2.11	2.11	
L-Trp	0.24	0.24	0.24	0.24	0.24	0.24	0.24	0.24	0.24	
Choline chloride	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	
Inert	1.29	2.29	0.45	0.76	0.00	0.44	2.93	0.00	2.87	
Sodium bicarbonate	0.84	0.84	0.84	0.84	0.84	0.84	0.84	0.84	0.84	
Premix Vitamin2	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	1.30	
Premix Mineral3	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	
Salinomycin, 12%	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50	
Antioxidant	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	0.20	
Avilamycin, 10%	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	
Calculated analysis (g per kg of diet)	
AME (MJ per kg)	12.45	12.45	12.45	12.45	12.45	12.45	12.45	12.45	12.45	
Available Ca	9.71	9.71	9.71	9.71	9.71	9.71	9.71	9.71	9.71	
Available P	4.63	4.63	4.63	4.63	4.63	4.63	4.63	4.63	4.63	
Na	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	2.00	
K	7.51	7.51	7.50	7.50	7.51	7.51	7.51	7.50	7.50	
Cl	2.91	2.91	2.90	2.90	2.91	2.91	2.91	0.290	2.90	
Digestible Lys	13.07	13.07	13.07	13.07	13.07	13.07	13.07	13.07	13.07	
Digestible Arg	12.60	15.37	12.60	15.37	12.02	15.95	13.98	13.98	13.98	
Digestible Gly+Ser	17.32	17.32	21.11	21.11	19.21	19.21	16.53	21.89	19.21	
Digestible Ser	8.82	8.82	8.82	8.82	8.82	8.82	8.82	8.82	8.82	
Glycine equivalents4	14.80	14.80	18.59	18.59	16.69	16.69	14.01	19.37	16.69	
Digestible Met+Cys	9.67	9.67	9.67	9.67	9.67	9.67	9.67	9.67	9.67	
Digestible Thr	8.63	8.63	8.63	8.63	8.63	8.63	8.63	8.63	8.63	
Digestible Trp	2.35	2.35	2.35	2.35	2.35	2.35	2.35	2.35	2.35	
Electrolytic balance (mEq per kg)	197	197	197	197	197	197	197	197	197	
Analyzed nutrients (g per kg of diet)	
Dry matter	878.2	871.1	877.6	874.8	877.5	877.7	878.9	871.7	876.6	
Crude protein	210.2	213.8	212.7	212.7	211.1	216.8	210.8	215.5	212.3	
Ether extract	46.62	49.17	47.59	48.91	47.73	44.88	46.72	47.56	48.11	
1 Arginine and glycine percentage compared to the amount of lysine proposed by Rostagno et al. (2017) for broilers.

2 Vitamin Premix (g per kg of diet): 1.35 mg vitamin (vit.) A; 31.25 mcg vit. D3; 14.9 mg vit. E; 1.25 mg vit. K3; 0.75 mg vit. B1; 3 mg vit. B2; 1.5 mg vit. B6; 6 mg vit. B12; 12.5 mg niacin; 6 mg pantothenic acid; 400 mcg folic acid; 30 mcg biotin; 125 mcg selenium.

3 Premix mineral (g per kg of diet): 10 mg Cu; 50 mg Fe; 80 mg Mn; 1 mg Co; 1 mg I; 50 mg Zn.

4 Glycine equivalents = glycine (g per kg) + (0.7143*serine (g per kg)).

For the phases from 11 to 21, 22 to 35 and 36 to 42 d, isocaloric and isoproteic diets (control) were formulated based on corn and soybean meal according to the feed composition and nutritional requirements of Lar Agroindustrial Cooperative (Table 2).Table 2 Ingredients and nutritional composition (g per kg, as-fed basis) used in commercial diet starter (d 11–21), grower (d 22–35) and withdrawal phases (d 35–42).

Table 2Ingredients	Grower	Finisher	Withdrawal	
Corn	510.9	599.1	608.9	
Soybean meal	393.0	323.5	315.5	
Soybean oil	51.30	37.20	39.90	
Mono-dicalcium phosphate	12.63	11.05	8.60	
Limestone	13.00	11.50	10.50	
NaCl	3.30	3.10	3.30	
DL-Met	3.95	3.85	3.60	
L-Thr	0.85	1.00	0.75	
Lys sulfate, 54.6%	3.50	4.15	3.40	
Choline chloride	0.50	-	-	
Sodium bicarbonate	2.00	1.50	1.50	
Premix1	4.482	3.433	3.434	
Protease5* + carbohydrases6	0.44	0.44	0.44	
Carbohydrase7	0.13	0.13	0.13	
Phytase8	0.05	0.05	0.05	
Calculated analysis (g per kg of diet)				
AME (MJ per kg)	12.77	13.19	13.40	
Available Ca	9.95	8.93	8.07	
Available P	4.90	4.50	4.00	
Na	2.02	1.81	1.88	
K	9.35	8.22	8.08	
Cl	2.56	2.38	2.51	
Digestible Lys	13.19	11.99	11.32	
Digestible Arg	14.17	12.33	12.16	
Digestible Met+Cys	10.20	9.51	9.12	
Digestible Met	6.95	6.53	6.23	
Digestible Thr	8.49	7.69	7.32	
Digestible Trp	2.40	2.09	2.05	
Digestible Val	9.40	8.40	8.24	
Electrolytic balance (mEq per kg)	249.61	221.47	218.04	
Analyzed nutrients (g per kg of diet)				
Dry matter	873.2	881.2	877.0	
Crude protein	215.2	207.7	200.5	
Ether extract	75.4	63.7	66.6	
1 Vitamin, mineral and enzymatic premix (dsm-firmenich, SP, BR).

2 Premix for starter phase (g per kg of diet): 2.42 mg vitamin (vit.) A; 89.6 mcg vit. D3; 40.05 mg vit. E; 2.69 mg vit. K3; 1.79 mg vit. B1; 6.27 mg vit. B2; 3.58 mg vit. B6; 13.44 mg vit. B12; 44.8 mg niacin; 10.75 mg pantothenic acid; 2.69 mg folic acid; 179.2 mcg biotin; 0.92 mg Cu; 5.5 mg Fe; 8.25 mg Mn; 99.5 mcg Co; 0.23 mg I; 9 mg Zn; 287.62 mg Se; 179 mg RONOZYME VP; 148 mg RONOZYME ProAct; 97 mg RONOZYME HiStarch; 35.84 mg RONOZYME HiPhos 2000; 35.84 mg nicarbazin; 44.8 antioxidant; 71.68 silicon dioxide.

3 Premix for grower phase (g per kg of diet): 2.32 mg vitamin (vit.) A; 85.75 mcg vit. D3; 38.33 mg vit. E; 2.57 mg vit. K3; 1.72 mg vit. B1; 6 mg vit. B2; 3.43 mg vit. B6; 12.86 mg vit. B12; 42.88 mg niacin; 10.29 mg pantothenic acid; 2.57 mg folic acid; 171.5 mcg biotin; 1.25 mg Cu; 7.5 mg Fe; 11.25 mg Mn; 125 mcg Co; 312.5 mcg I; 12.5 mg Zn; 300 mg Se; 205.8 mg RONOZYME VP; 171.5 mg RONOZYME ProAct; 111.5 mg RONOZYME® HiStarch; 78.18 mg RONOZYME HiPhos 2000; 77.18 mg lasalocid; 34.3 antioxidant.

4 Premix for finisher phase (g per kg of diet): 1.16 mg vitamin (vit.) A; 300 mcg vit. D3; 38.33 mg vit. E; 1.29 mg vit. K3; 0.857 mg vit. B1; 3 mg vit. B2; 1.72 mg vit. B6; 6.43 mg vit. B12; 21.44 mg niacin; 5.15 mg pantothenic acid; 1.29 mg folic acid; 85.75 mcg biotin; 1 mg Cu; 6 mg Fe; 9 mg Mn; 1 mcg Co; 250 mcg I; 10 mg Zn; 214.38 mg Se; 205.8 mg RONOZYME VP; 171.5 mg RONOZYME ProAct; 111.48 mg RONOZYME HiStarch; 34.3 antioxidant.

5 RONOZYME ProAct

6 RONOZYME VP

7 RONOZYME HiStarch

8 RONOZYME HiPhos.

*The AA matrix was improved by 3% with the addition of protease.

Broiler chickens were housed in an aviary with exhaust fans, and evaporative cooling cell system. Pens had 1.96 m2 with concrete floor covered with used (4 times) wood shavings and a housing density of 12.75 broiler chickens per m2. Animals received water and feed ad libitum. The temperature control and the lighting program followed the guidelines outlined in the breeder's manual for each growing phase.

Growth Performance

On the day of placement (d 1 post-hatch), all birds in each pen were weighed collectively using a calibrated precise scale (WBW/Adam Equipment, Maidstone, UK). On d 10, 21, and 42, the birds and remaining feed were weighed by pen to determine the body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR). Mortality was recorded daily and categorized by treatment. Furthermore, mortality was used for FI and BWG corrections, according to the methodology proposed by Sakomura and Rostagno (2016).

Blood Analysis

At d 42, after 6 h of fasting, 2 broilers per pen were randomly selected for blood collection by ulnar punction. Samples were kept at rest for 15 min at room temperature and then centrifuged at 1,050 g for 10 min (Kasvi K14-4000, Kasvi, São Paulo, BR) in order to obtain serum for the biochemical blood profile analysis.

The time between sample collection and storage was approximately 30 min. The serum samples were stored at frozen conditions (-20 C°) for approximately 30 d until further analysis (Nunes et al., 2018). Posteriorly, serum samples were thawed at room temperature and the blood constituents glucose (GLU), cholesterol (CHO), triacylglycerol (TLG), total protein (TP), albumin (ALB), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), creatine kinase (CK), and gamma-glutamil transferase (GGT) were determined using commercial kits (Elitech, Clinical Systems, Elitech Group, Paris, FR) in a spectrophotometer with automatic calibration and high-performance analyzer (Flexor EL200, ELITech Latin America, Brazil).

Proximate Composition of Pectoralis Major Muscle

The evaluation of the proximate composition of the breast muscle (Pectoralis major) was performed using the right side of the breast from 1 randomly selected broiler per pen. Breast samples were pre-dried in a forced air circulation oven at ± 55°C for 72 h. Posteriorly, samples were analyzed for crude protein (CP) (AOAC, 2010, method 968.06), dry matter (DM) (AOAC, 2006, method 934.01), ether extract (EE) (Association of Official, 2000, method 960.39), and hydroxyproline (Association of Official, 2000, method 990,26).

Incidence of Myopathies

For the evaluation of the incidence of myopathies in the breast muscle, 15 broilers per pen (n = 1,680) were selected and slaughtered at the Poultry and Meat Industrial Unit of Lar Cooperative.

Wooden Breast scores were classified according to Tijare et al. (2016) as: Normal (score 0): without any area of hardness or paleness. Mild (score 1): hard mainly in the cranial and caudal region but flexible otherwise. Moderate (score 2): moderately affected throughout the muscle. Severe (score 3): affected throughout the muscle with surface bleeding and the presence of a sterile exudate.

For the evaluation of White Stripping severity, striations were adapted from Kuttappan et al., (2012) and classified as: Normal (score 0): breast fillets without visible white striations. Mild (score 1): white lines, usually < 1 mm thick, but visible on the fillet surface. Moderate (score 2): large white lines (1–2 mm thick) clearly visible on the fillet surface, covering less than 50% of the fillet. Severe (score 3): whitish stripes, parallel to the muscle fiber, >2 mm thick, covering almost the entire fillet surface).

Statistical Procedures

Statistical analysis was performed using the software SAS University Edition (2017) (SAS Inst. Inc., Cary, NC). Data were tested for normality (Shapiro–Wilk), using the procedure UNIVARIATE.

The statistical model used was:Yijkn=μ+Ti+bj+Pk+T*b*Pijk+eijkn

Where: Yijkn: observation related to i-th Arg concentration, j-th Gly+Ser concentration, k-th GAA class in n-th repetition; µ: mean; Ti: effect corresponding to i-th Arg concentration; βi: effect corresponding to j-th Gly+Ser concentration; Pk: effect corresponding to k-th GAA class; T*β*Pijk: Interaction effect between the i-th T, j-th β and k-th P; eijkn: residual error associated to i-th T, j-th β and k-th P in n-th repetition.

The data were submitted to variance analysis (ANOVA), considering the isolated effects and the three components between the factors studied, Arg, Gly+Ser concentrations and GAA classes.

Regression analysis was used to evaluate the isolated effects to estimate Arg and Gly+Ser concentrations using the procedure GLM and REG. Furthermore, Dunnett test was used to compare Arg and Gly+Ser concentrations to central point. The means between GAA classes were compared using the F test. The significance concentration adopted in all hypothesis tests was α ≤ 0.05. Pearson's correlation coefficient was employed using CORR procedure to determine any correlation between aspartate aminotransferase and creatine kinase data.

Data on the incidence of myopathies for each bird in the same pen was grouped, thus the proportion of each score within the pen was calculated. For the variables normal and severe score for WB, and moderate and moderate+severe score for WS, the GLIMMIX was adjusted to the data using the Inverse Gaussian distribution and Log binding function. For the variables mild, moderate, moderate+severe scores for WB, and mild score and mean scores for WS, the GLIMMIX was adjusted to the data using the Gamma distribution and Log binding function. For the variables normal and severe score for WS, the GLIMMIX was adjusted to the data, using the Tweedie distribution and Log binding function. The Akaike Information Criterion (AIC) together the graphic analysis of residue adhesion was used as criteria to determine the quality of adjustment of the model.

RESULTS

Growth Performance

No significant effect (P > 0.05) of the three components between digestible Arg, Gly+Ser, and GAA concentrations was observed on broilers performance (d 1–10, d 1–21, and d 1–42) for any of the evaluated periods (Table 3). From d 1 to 10, based on the F test, there was an improvement on FCR (P = 0.0481) where the group of animals that were fed GAA presented reduced FCR compared to those without GAA (1.161 vs. 1.175).Table 3 Performance of broilers fed concentrations of Arg and Gly up to d 10, supplemented or not with GAA.

Table 3	BWG	FI	FCR	
Arginine1	d0-10	d0-21	d0-42	d0-10	d0-21	d0-42	d0-10	d0-21	d0-42	
 92	259	1096	3349	309	1331	4984	1.197	1.215	1.489	
 96.4	258	1090	3346	305	1332	4939	1.179	1.213	1.477	
 107	261	1096	3325	305	1328	4910	1.166	1.212	1.476	
 117.6	263	1093	3320	304	1323	4880	1.154	1.211	1.470	
 122	264	1111	3383	305	1341	4983	1.158	1.208	1.473	
Glycine2										
 126.5	259	1096	3344	310	1334	4951	1.195	1.218	1.480	
 132.5	262	1088	3349	311	1323	4931	1.186	1.217	1.472	
 147	262	1100	3332	305	1331	4930	1.164	1.21	1.480	
 161.5	259	1096	3317	298	1321	4888	1.147	1.206	1.474	
 167.5	257	1083	3324	303	1322	4851	1.179	1.221	1.459*	
Additive3										
 Without GAA	261	1094	3328	306	1330	4933	1.179a	1.216	1.482a	
 With GAA	262	1098	3336	304	1326	4908	1.161b	1.208	1.470b	
 SEM	0.866	2.406	10.51	1.069	2.017	13.97	0.004	0.003	0.002	
P-values										
 Arg	0.306	0.394	0.475	0.671	0.801	0.285	0.013	0.693	0.318	
 Gly	0.324	0.165	0.758	0.323	0.276	0.388	0.001	0.593	0.049	
 GAA (Ga)	0.216	0.418	0.874	0.843	0.657	0.477	0.048	0.693	0.026	
 A*G*Ga	0.216	0.283	0.621	0.642	0.504	0.563	0.178	0.123	0.985	
Regression										
 Arg							0.009 (L)			
 Gly							0.047 (Q)		0.132	
1 Percentage of arginine in relation to diet lysine;

2 Percentage of glycine+serine in relation to diet lysine;

3 GAA: guanidinoacetic acid ; L: linear effect; Q: quadratic effect; SEM: standard error of the mean.

FCR (d 10) = 1.276591975 – 0.001011044*Arg; R2= 0.46; FCR (d 10) = 2.361708041 – 0.015521714*Gly+Ser + 0.00004993*Gly+Ser2; R2= 0.39; % of Gly+Ser by the 1st derivation = 155.44; Estimated response of FCR = 1.155 (g per g).

⁎ Averages followed by * differ from Gly “147%” inclusion concentration by the Dunnett test, at a 5% probability concentration.

Furthermore, the FCR from d 1 to 10 showed a decreasing linear response (P = 0.013) as a function of Arg concentrations, as well as a quadratic response (P = 0.001) depending on Gly+Ser concentrations, with a minimum response predicted at 155.44% (Figure 1). No differences (P > 0.05) were observed for BWG, FI, and FCR between Arg, Gly+Ser or GAA supplementation during the period from d 1 to 21 (Figure 2).Figure 1 FCR0-10d = 3.1226 – 0.0125*Arg - 0,0158*Gly +0.00004*Arg2 + 0.00005*Gly2 + 0.00001; Significant values are in bold (P < 0.05); Trends are showed with underlined values (P = 0.10).

Figure 1

Figure 2 FCR0-21d = 1.1057 + 0.087*Arg - 0,0045*Gly - 0.00002*Arg2 + 0.00002*Gly2 - 0.00003; Significant values are in bold (P < 0.05); Trends are showed with underlined values (P = 0.10).

Figure 2

Considering the period from 1 to 42 d, broilers fed GAA presented improved FCR (P = 0.026) compared to those fed diet without GAA supplementation (1.470 vs. 1.482) (Figure 3). The FCR was influenced (P = 0.049) by Gly+Ser concentrations; however, the data did not fit to any regression model (P > 0.05). Considering the Dunnett's test, the concentration of 167.5% of digestible Gly+Ser presented differences (P < 0.05) in relation to the central point (147% of Gly+Ser), presenting an improved FCR (1.459 vs. 1.480). No differences were observed (P > 0.05) on BWG and FI when considering the concentrations of digestible Arg, Gly+Ser, and GAA supplementation.Figure 3 FCR0-21d = 1.3624 - 0.043*Arg + 0,0053*Gly + 0.00002*Arg2 - 0.00002*Gly2 - 0.00002; Significant values are in bold (P < 0.05); Trends are showed with underlined values (P = 0.10).

Figure 3

Blood Analysis

The analysis of energy, protein (Table 4), and enzymatic serum biochemical parameters (Table 5) revealed no significant effects attributed to either the 3 components or treatments (P > 0.05). However, there was a significant effect (P = 0.028) on the concentration of AST by the F test for GAA supplementation, where broilers fed diet with GAA showed an increase in AST concentration compared to the non-supplemented group (Figure 4). Also, AST concentration displayed an increasing (P = 0.008) and a decreasing (P = 0.020) linear effect as a function of digestible Arg and Gly+Ser concentrations, respectively. No interaction (P > 0.05) between digestible Arg, Gly+Ser and GAA classes was observed for the other enzymatic biochemical parameters.Table 4 Biochemical parameters of serum of broilers at d 42 of age fed diets with concentrations of Arg and Gly up to d 10, supplemented or not with GAA.

Table 4Arginine1	Glucose (mg/dL)	CHO (mg/dL)	TGL (mg/dL)	TP (g/l)	ALB (g/L)	Glob (g/L)	UA (mg/dl)	
 92	253	147	37.50	27.42	15.04	12.38	2062	
 96.4	245	152	38.94	27.30	14.71	12.59	2573	
 107	255	152	37.67	27.74	15.33	12.40	2529	
 117.6	251	161	40.69	29.19	15.75	13.44	2733	
 122	253	142	42.50	27.766	14.86	12.90	2103	
Glycine2								
 126.5	258	160	32.87	29.32	16.46	12.85	2404	
 132.5	251	160	39.94	28.09	15.51	12.57	2617	
 147	254	149	38.77	27.53	15.09	12.44	2468	
 161.5	245	153	39.69	28.40	14.95	13.45	2689	
 167.5	253	151	38.37	27.49	15.34	12.15	2251	
Additive3								
 Without GAA	254	153	39.34	28.10	15.36	12.74	2511	
 With GAA	252	151	37.89	27.62	15.14	12.48	2490	
 SEM	1.771	2.520	1.325	0.273	0.250	0.303	136.2	
P-values								
 Arg	0.940	0.655	0.867	0.327	0.755	0.879	0.695	
 Gly	0.814	0.712	0.807	0.425	0.602	0.8674	0.934	
 GAA (Ga)	0.950	0.720	0.849	0.743	0.943	0.825	0.905	
 A*G*Ga	0.964	0.893	0.962	0.418	0.734	0.680	0.593	
1 Percentage of arginine in relation to diet lysine;

2 Percentage of glycine+serine in relation to diet lysine;

3 GAA: guanidinoacetic acid; CHO: cholesterol; TGL: triglycerides; TP: total protein; ALB: albumin; Glob: globulin; UA: uric acid; SEM: standard error of the mean.

Table 5 Enzymatic serological profile of broilers at d 42 of age fed diets with concentrations of Arg and Gly up to d 10, supplemented or not with GAA.

Table 5Arginine1	ALT (IU/L)	AST (IU/L)	CRE (mg/dL)	CK (U/L)	GGT (IU/L)	
 92	7.67	369	0.10	27546	19.74	
 96.4	9.23	438	0.10	30144	20.69	
 107	8.29	456	0.11	33338	22.62	
 117.6	8.47	491	0.11	36423	25.68	
 122	8.45	514	0.12	41389	25.47	
Glycine2						
 126.5	8.42	506	0.12	33194	22.82	
 132.5	9.76	482	0.11	36008	22.67	
 147	8.30	458	0.11	34014	23.07	
 161.5	7.94	448	0.11	30587	23.70	
 167.5	7.57	365	0.11	30628	19.23	
Additive3						
 Without GAA	8.68	475a	0.11	34.225	22.34	
 With GAA	8.16	438b	0.11	32.903	23.29	
SEM	0.411	11.086	0.003	1317	0.132	
P-values						
 Arg	0.932	0.026	0.468	0.182	0.182	
 Gly	0.655	0.042	0.626	0.701	0.621	
 GAA (Ga)	0.227	0.028	0.511	0.146	0.802	
 A*G*Ga	0.623	0.666	0.548	0.879	0.186	
Regression						
 Arg		0.008 (L)				
 Gly		0.020 (L)				
1 Percentage of arginine in relation to diet lysine;

2 Percentage of glycine+serine in relation to diet lysine

3 GAA, guanidinoacetic acid; L: linear effect; ALT: alanine aminotransferase; AST: aspartate aminotransferase; CRE: creatinine; CK: creatine kinase; GGT: gamma-glutamyl transferase; SEM: standard error of the mean.

AST = 21.8323604 + 4.03801532*Arg; R2= 0.25; AST = 846.3446535 – 2.683467* Gly; R2 = 0.21.

Figure 4 AST = -3,578.4230 + 38.2811*Arg + 26.7765*Gly - 0.04*Arg2 - 0.0344*Gly2 - 0.1773; Significant values are in bold (P < 0.05); Trends are showed with underlined values (P = 0.10).

Figure 4

Proximate Composition of Pectoralis Major Muscle

No effects of the 3 component (P > 0.05) on the proximate composition (crude protein, dry matter, crude fat, and collagen) of breast meat was observed (Table 6). Crude fat presented a quadratic response (P = 0.015) as a function of digestible Gly concentrations, with a minimum response predicted at 154.9% concentration (Figure 5).Table 6 Proximate composition (g per kg) of broiler breast at d 42 of age fed diets whith concentrations of Arg and Gly up to d 10, supplemented or not with GAA.

Table 6Arginine1	Crude protein	Moisture	Fat	Collagen (mg per g)	
 92	201.5	749.7	13.8	494	
 96.4	201.8	751.1	17.2	472	
 107	197.7	752.4	15.9	469.3	
 117.6	201.9	749.6	15.2	467	
 122	193.6	761.9	16.8	456.1	
Glycine2					
 126.5	193.2	753.9	19.2	489.9	
 132.5	198.8	749.2	18.3	457.1	
 147	198.1	752.9	15.1	474.9	
 161.5	204.9	751.5	14.1	481.9	
 167.5	199.3	754.1	16.6	417.7	
Additive3					
 Without GAA	198.6	751.5	16.6	461.4	
 With GAA	199.5	753.1	15.2	479.3	
 SEM	1.396	1.449	0.557	1.103	
P-values					
 Arg	0.749	0.334	0.565	0.932	
 Gly	0.746	0.938	0.033	0.519	
 GAA (Ga)	0.497	0.557	0.482	0.417	
 A*G*Ga	0.287	0.183	0.503	0.925	
Regression					
 Gly	-	-	0.015 (Q)	-	
1 Percentage of arginine in relation to diet lysine.

2 Percentage of glycine+serine in relation to diet lysine.

3 Guanidinoacetic acid (GAA); Q= Quadratic effect; SEM: Standard error of the mean.

Fat = 17.821899 – 0.21128073*Gly + 0.00068196*Gly2; R2 = 0.19; % of Gly by the 1st derivation = 154.91; Estimated response of fat = 1.45 %.

Figure 5 Fat = 114.2747 + 0.5274*Arg – 1.6303*Gly + 0.0008*Arg2 + 0.0069*Gly2 - 0.0048; Significant values are in bold (P < 0.05); Trends are showed with underlined values (P = 0.10).

Figure 5

Incidence of Myopathies

No interaction (P > 0.05) was observed between the concentrations of digestible Arg, Gly+Ser, and GAA supplementation regarding the proportion of each WB and WS scores, as well as their average scores (Table 7).Table 7 Proportions of occurrence and mean scores for Wooden Breast in broiler breast filets at d 42 of age fed diets with concentrations of Arg and Gly up to d 10, with or without supplemental GAA.

Table 7Arginine1	Occurrence of scores (%)	Means of Scores	
Normal	Mild	Moderate	Severe	Mod+Sev	
 92	45.86	22.23	18.00	13.92	31.91	1.16	
 96.4	37.90	24.10	27.44	10.56	38.00	1.10	
 107	41.09	23.44	22.91	12.56	35.47	1.06	
 117.6	48.64	21.71	20.11	9.54	29.64	0.91	
 122	31.68	30.03	26.82	11.47	38.29	1.18	
Glycine2							
 126.5	40.90	25.18	24.20	9.72	33.91	1.03	
 132.5	40.94	21.06	27.48	10.52	38.01	1.08	
 147	40.69	23.72	22.19	13.40	35.59	1.10	
 161.5	45.60	24.76	20.06	9.58	29.64	0.93	
 167.5	39.85	24.87	26.38	8.90	35.28	1.04	
Additive3							
 Without GAA	41.43	20.54b	25.27	12.77	38.04a	1.10	
 With GAA	41.34	26.80a	20.90	10.95	31.86b	1.02	
 SEM	1.346	1.158	1.091	0.843	1.315	0.028	
P-values							
 Arg	0.1267	0.6486	0.1584	0.6245	0.3402	0.1783	
 Gly	0.865	0.799	0.275	0.487	0.372	0.391	
 GAA (Ga)	0.613	0.015	0.445	0.495	0.039	0.362	
 A*G*Ga	0.181	0.243	0.274	0.131	0.163	0.133	
1 Percentage of arginine in relation to diet lysine;

2 Percentage of glycine+serine in relation to diet lysine;

3 Guanidinoacetic acid (GAA); Added to the most severe moderate scores averages (Mod+Sev); SEM: Standard error of the mean.

The supplementation of GAA significantly influenced (P = 0.015) the occurrence of mild WB score. Broilers fed GAA displayed a higher proportion of mild score (26.80 vs. 20.54%) compared to those fed non-supplemented diet. Additionally, broilers on GAA diet exhibited reduced sum of moderate+severe scores (P = 0.039) compared to the non-supplemented group (31.86 vs. 38.04 %).

There was no effects of the 3 components or isolated effect of digestible Arg and Gly+Ser concentrations (P > 0.05) on the proportions and average scores of WS (Table 8). The GAA supplementation affected the proportion of normal (P = 0.002) and severe (P = 0.029) WS scores occurrence. Broilers fed diets containing GAA had more normal scores (10.27 vs. 8.48%; P = 0.002) and a reduction in breast filets classified as severe (5.80 vs. 7.59%; P = 0.029) compared with those not receiving GAA.Table 8 Proportions of occurrence and mean scores for White Striping in broiler breasts at d 42 of age fed concentrations of Arg and Gly up to d 10, supplemented or not with GAA.

Table 8	Occurrence of scores (%)	Means of Scores	
Arginine1	Normal	Mild	Moderate	Severe	Mod+Sev	
 92	15.63	56.25	21.88	6.25	28.13	1.19	
 96.4	6.25	64.06	20.31	9.38	29.69	1.31	
 107	7.42	60.94	25.78	5.08	30.86	1.27	
 117.6	15.63	62.50	15.63	6.25	21.88	1.13	
 122	12.50	43.75	28.13	15.63	43.75	1.47	
Glycine2							
 126.5	9.38	59.38	28.13	3.13	31.25	1.25	
 132.5	14.06	64.06	17.19	4.69	21.88	1.11	
 147	9.38	57.81	25.78	6.25	32.03	1.29	
 161.5	7.81	62.50	18.75	10.94	29.69	1.33	
 167.5	3.13	65.63	21.88	9.38	31.25	1.22	
Additive3							
 Without GAA	8.48b	59.82	23.21	7.59a	30.80	1.28	
 With GAA	10.27a	60.27	23.66	5.80b	29.46	1.25	
 SEM	1.350	2.275	1.829	1.051	2.083	0.032	
P-values							
 Arg	0.619	0.4621	0.148	0.151	0.853	0.130	
 Gly	0.150	0.9509	0.289	0.119	0.415	0.241	
 GAA (Ga)	0.002	0.572	0.306	0.029	0.174	0.638	
 A*G*Ga	0.389	0.945	0.092	0.892	0.446	0.710	
1 Percentage of arginine in relation to diet lysine;

2 Percentage of glycine+serine in relation to diet lysine;

3 GAA: guanidinoacetic acid; Added to the most severe moderate scores averages (Mod+Sev); SEM: standard error of the mean.

DISCUSSION

In the current study, it was observed that while BWG and FI remained unaffected by GAA supplementation, regardless of the amino acid concentration, there was a reduction in FCR from d 1 to 10 and d 1 to 42 in broilers receiving GAA. This improvement in FCR, with no changes in FI, suggests an increase in energy efficiency resulting from GAA supplementation into the diet (Ahmadipour et al. 2018). This finding is consistent with several previous studies that also reported enhanced FCR with GAA supplementation (Ahmadipour et al., 2018; Córdova-Noboa et al., 2018; He et al., 2019; DeGroot et al., 2019; Souza et al., 2021). Michiels et al. (2012) documented the beneficial impact of GAA supplementation, particularly during the final phase of development, notably improving the gain:feed ratio. The well-documented improvements in FCR may be attributed to GAA supplementation resulting in reduced caloric intake per kg of BW and carcass weight, thereby leading to a decrease in FCR (Mousavi et al., 2013).

Elevated concentrations of CRE, ATP, and phosphocreatine (PCRE), as well as an augmented PCRE to ATP ratio, have been reported in the breast meat of broiler chickens following GAA supplementation (Michiels et al., 2012; Tossenberger et al., 2016; Yazdi et al., 2017; DeGroot et al., 2018). Furthermore, these factors, integral to energy metabolism, play crucial roles in various biochemical processes such as ion transport, muscle contraction, nerve impulse propagation, substrate phosphorylation, and chemical synthesis (Bonora et al., 2012). The increase in available energy, driven by ATP, can potentially enhance these processes (Ale Saheb Fosoul et al., 2018).

It is important to be cautious when comparing results across various published studies aimed at evaluating dietary Arg supplementation in broiler chickens. Notable differences include strain, gender, age, number and duration of feeding phases, basal diet composition, and Arg inclusion concentration (Wu et al., 2011).

Regarding the productive aspects, it was observed that broilers receiving a diet with a digestible Arg:Lys ratio of 122% displayed reduction in FCR from d 1 to 10 of age, suggesting the crucial role of Arg in early life stages (Corzo and Kidd, 2003; Dao and Swick, 2021). Broilers have a high requirement for Arg due to their inability to produce essential enzymes necessary for the novo synthesis of this amino acid. Additionally, they lack the ability for citrulline production in the small intestine and exhibit high renal arginase activity (Tamir and Ratner, 1963; Wu et al., 1995; Bortoluzzi et al., 2018). Furthermore, Arg requirements are influenced by environmental factors such as housing conditions, including ambient temperature and altitude, as well as dietary factors such as protein concentration, protein sources, and the balance between AA, along with other stressful conditions (Khajali and Wideman, 2010; Kodambashi Emami et al., 2017).

In addition to its function as a protein constituent, Arg serves as a precursor for the synthesis of creatine, polyamines, nitric oxide, and it may stimulate the secretion of insulin-like growth factor hormone (Fernandes and Murakami, 2010), while also influencing the development of lymphoid organs and bones, playing an important role in the defense system (Dao and Swick, 2021). These mechanisms of action may help explain the positive effects of Arg supplementation on performance and small intestinal morphology of 1-wk-old broilers (Murakami et al., 2012).

The results in the present study align with findings by Zampiga et al. (2018), who observed an improvement in the FCR of broiler chickens from d 1 to 12 when supplemented with a digestible Arg:Lys ratio of 115%, compared to birds that received the ratio recommended of 105% by the NRC. These authors concluded that the ratio digestible Arg:Lys recommended for diets without the inclusion of animal by-products may be underestimated to achieve maximum performance in modern broiler chickens.

In a study by Ebrahimi et al. (2014), an improvement in feed efficiency was documented at d 10, 24, and 46, correlating with an increase in digestible Arg concentration from the 100% recommended for Ross broiler chickens (Arg:Lys of 108%, 13.1 g per kg) to 153%, 168%, and 183%. Additionally, Murakami et al. (2012) observed a linear correlation between digestible Arg concentrations and FCR in broiler chickens from 1 to 7 d, with birds receiving a concentration of 17.9 g per kg (Arg:Lys of 142%) demonstrating improved FCR. In a study by Laika and Jahanian (2017), no difference was observed in the FCR of broilers fed diets with the addition of digestible Arg to meet 100% or exceed NRC recommendations (105 e 110%). Furthermore, the results obtained in the present study suggest that the digestible Arg:Lys ratio (107%), may be insufficient to achieve the maximum productive potential of chickens.

The optimization of digestible Gly+Ser in the diet from d 1 to 10 was achieved with a Gly+Ser:Lys ratio of 156.26% (20.4 g per kg) for FCR. Current Brazilian recommendations suggest 19.21 g per kg for digestible Gly+Ser and a Gly+Ser:Lys ratio of 147% for average performance in male broiler chicken from d 1 to 7 (Rostagno et al., 2017). Similarly, Ospina-Rojas et al. (2013) recommended 21.2 and 21.6g per kg of digestible Gly+Ser to obtain the best BWG and FCR for broilers from 1 to 7 d, respectively, representing a digestible Gly+Ser:Lys ratio of 154.7 and 157.7%. The authors observed that the demands of digestible Gly+Ser are lower when the diet is supplemented with threonine (Ospina-Rojas et al., 2013; Rostagno et al., 2017).

There is a substantial variation in the estimated requirements for digestible Gly+Ser among studies. Dean et al. (2006) attribute these discrepancies to a multitude of compounds that can be converted into Gly, as well as the use of different protein sources and concentrations of compounds involved in Gly metabolism between experimental diets, strain, gender, and age of birds. According to Hofmann et al. (2019), the requirements for digestible Gly+Ser are influenced by dietary crude protein, which is one of the main factors that effects the requirement for Gly.

The enzyme AST catalyzes the transfer of the amino group from aspartate to α-ketoglutarate, resulting in the production of oxaloacetate and glutamate (Adeva et al., 2012). It is present in mitochondria and cytoplasm, existing in different forms (Burtis and Bruns, 2014). This enzyme is found in various tissues, with higher concentrations in the liver, erythrocytes, heart, and skeletal muscles (Gonzáles and Silva, 2017). Aspartate aminotransferase is a sensitive indicator of liver diseases. However, because this enzyme is present not only in liver tissues but also in all types of muscles, an increase in its activity commonly indicates damage not only to the liver but also to the muscles (Hochleithner and Hochleithner, 2006).

In this context, an increase in AST activity should be interpreted along with creatine kinase (CK), a specific enzyme of the muscle. Simultaneous increases in CK and AST activity suggests that the elevated AST concentration is likely due to muscle damage. However, because AST has a longer plasma half-life than CK, an increase in its concentration, even without the elevation of CK activity, may also indicate muscle damage (Jaensch, 2000; Hochleithner and Hochleithner, 2006).

Although CK was not influenced by treatments in the present study, a positive correlation of 0.81 between CK and AST was observed. Based on these results, it can be inferred that the increase in digestible Arg:Lys ratio up to 122% may indicate muscle damage due to the rise in AST activity. Conversely, the digestible Gly+Ser:Lys ratio and GAA diet supplementation reduced the concentration of this enzyme, suggesting a reduction in muscle damage and a protective role of this additive.

In a study by Ebrahimi et al. (2014), CP and DM content of breast meat showed an increase in response to dietary supplementation of 153% digestible Arg, compared to a diet based on Ross recommendations (13,1, 12,1 and 10,4 g per kg of digestible Arg, for the initial/starter phases, grower, and termination/final phases, respectively). These authors also reported that significant changes in the content of ash and fat require higher concentrations of digestible Arg (183% and 168%, respectively).

Considering the results of the present study, the dietary supplementation of GAA increased the proportions of normal and mild scores of WS and WB in the breast muscle, while providing a reduction in the severe score of WS and the sum of moderate+severe WB scores.

The exact etiology of WB and WS myopathies has not been fully elucidated, nor has their relationship with each other. Studies have shown that breasts affected by these myopathies exhibit characteristics such as severe hypoxia, oxidative stress, reduced glycolytic metabolites and muscle glycogen, profound alterations in inflammatory status, calcium signaling pathways, citrulline synthesis, and nitric oxide (NO) production from Arg (Mutryn et al., 2015; Russo et al., 2015; Zambonelli et al., 2016; Abasht et al., 2016; Boerboom et al., 2018; Soglia et al., 2020).

Another factor associated with the onset of myopathies is the increased accumulation rate of breast muscle, leading to hypertrophy of muscle fibers’ transverse area (Daughtry et al., 2017; Meloche et al., 2018; Lilburn et al., 2019) and reduced space for connective tissues (Christov et al., 2007; Sihvo et al., 2018; Velleman, 2019). This results in inadequate vascular growth, impairing oxygen supply, nutrient delivery, and removal of metabolic waste products from the muscle fibers of the breast (Zambonelli, et al., 2016; Boerboom et al., 2018; Lilburn et al., 2019).

Dietary supplementation of GAA has been shown to reduce the severity of myopathies in broilers fed plant-based diets, as evidenced by the evaluation of the blood parameters and the incidence of myopathies. The apparent improvement in the concentration of metabolites involved in muscle energy metabolism, such as CRE and glycogen content, due to GAA dietary supplementation (DeGroot et al., 2018), suggests a supportive effect on muscle energy metabolism (Kolling et al., 2013; Nabuurs et al., 2013).

Kolling et al. (2013) observed that CRE alters glucose oxidation, protecting the muscle from energy imbalances in hyperhomocystemic rats. Creatinine supplementation is indicated as adjuvant therapy to improve some clinical signs related to energy imbalance observed in patients affected by this disease. According to Córdova-Noboa et al. (2018), supplementation of GAA as a precursor of CRE can prevent or reduce the occurrence of WB by modulating the intermediate metabolites of muscle and energy metabolism.

Satellite cells (SC) are muscle-specific stem cells located at the periphery of muscle fibers, playing a crucial role in providing myogenic precursors for muscle fiber growth and repair (Moss and Leblond, 1970). These cells exhibit high activity post-hatch and proliferate to expand their population within the muscle. However, as birds age, the activity of these cells tends to decline (Daughtry et al., 2017).

Inadequate nutrition provided to newly hatched chicks can reduce activity and number of SC (Harthan et al., 2014; Powell et al., 2014). Therefore, it is crucial to ensure that the diet meets the requirements to stimulate early growth in chicks, promoting maximum proliferation and activity of SC to support muscle development (Bailey et al., 2020). Although we did not observe effects of AA concentrations on the incidence of myopathies, the primary goal of supplying adequate nutrition in the initial phase would be to promote the proliferation of SC.

A previous published study demonstrated that supplementation with 15 g per kg of Arg (increasing the digestible Arg:Lys ratio from 105%, 106%, and 107% to 120%, 115%, 110%, and 95% in the initial, growth I, growth II, and final phase, respectively) did not significantly affect the occurrence of WS, WB, and spaghetti meat (SM) in 43 d-old broiler chickens (Zampiga et al., 2018).

On the other hand, Bodle et al. (2018) observed a reduction in the average WB score in birds that received diets supplemented with digestible Arg (Arg:Lys ratio = 120%, 126%, and 141%) compared to those fed the control diet (Arg:Lys ratio = 112, 114, 115, 128%). This reduction may be attributed to the ability or Arg to improve blood oxygen flow, thereby reducing the hypoxic state of the muscle. However, no effect was observed for the incidence of WS.

In a study by Zampiga et al. (2019), an increase in digestible Arg:Lys ratios from 105%, 105%, 106%, and 107% (respectively in the initial, growth I, growth II, and final phase) in the control diet to 135%, 135%, 136%, and 137%, respectively, resulted in a reduction in the incidence and severity of WS and SM myopathies. However, no effect was observed on the incidence of WB.

Although the concentration of nitric oxide (NO) was not directly evaluated, Zampiga et al. (2019) hypothesized that the reduction in the incidence of myopathies may be associated with an increase in NO production via the Arg-NO pathway. Nitric oxide is a potent vasodilator that can enhance blood flow in the breast musculature, assisting in the supply of nutrients and removal of metabolic waste from muscle cells (Khajali and Wideman, 2010; Bloomer, et al., 2010).

These studies demonstrated the importance of nutritional strategies in potentially eliminating or significantly reducing the occurrence of myopathies. This is particularly significant given the low heritability observed for WB (0.02-0.1; 0.04-0.07) and the low to moderate heritability for WS (0.19-0.34; 0.25) (Bailey et al., 2015; Bailey et al., 2020).

Furthermore, these studies have reported low to moderate genetic correlations between myopathies and traits such as body weight and fillet yield, indicating that selection for these traits does not fully account for the manifestation of myopathies (Bailey et al., 2015; Bailey et al., 2020). The strategic reduction of amino acids used for maximum muscle deposition, such as digestible Lys, during specific growth phases has been shown to reduce the incidence of some myopathies (Cruz et al., 2017; Meloche et al., 2018c). Thus, further studies are warranted to evaluate the alterations in relationships among amino acids in the diet during different growth phases, in order to determine the most appropriate timing for implementing such strategies.

CONCLUSIONS

The current study demonstrates that optimizing the amino acid ratios and supplementing diets with GAA significantly improves the FCR throughout the entire growth period of broiler chickens. Specifically, a digestible Arg:Lys of 122% enhances the FCR during the starter phase (d 1–10), though it may be linked to increased muscle damage in later stages. Conversely, digestible Gly+Ser:Lys ratios of 156% and 167.5% notably enhance FCR across both the starter and overall growth periods, while also reducing breast meat fat content and potentially decreasing muscle damage.

Furthermore, the inclusion of 0.6 g per kg GAA in plant-based diets not only improves FCR but also significantly reduces the incidence of myopathies and mitigates muscle damage in broiler chickens. These findings highlight the importance of dietary optimization in enhancing growth performance and muscle health in poultry.

DISCLOSURES

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Brazil, for the scholarship granted to the first author during the doctoral program - Finance code 001.
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Further readings

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