Metabolizable energy equivalence of guanidinoacetic acid in corn soybean meal-based broiler diets

ABSTRACT In this study, we evaluated how guanidinoacetic acid (GAA) addition in diets with various metabolizable energy (ME) contents affects the performance of broiler chickens. We also estimated the equivalence of GAA in ME. We distributed 1,280 one-day-old broilers in a completely randomized design with eight treatments, eight replicates, and twenty birds per experimental unit. Treatments were based on ME levels (2,775-2,875-2,975 kcal/kg; 2,850-2,950-3,050 kcal/kg; 2,925-3,025-3,125 kcal/kg; or 3,000-3,100-3,200 kcal/kg, from 1 to 7, 8 to 21, and 22 to 42 days of age) and the inclusion of GAA (0 or 600 mg/kg). Supplementation of GAA increased weight gain in broilers at an energy level of 2,908 kcal/kg and improved feed conversion ratio (FCR) at energy levels of 2,908 and 2,983 kcal/kg. There was a linear reduction in feed intake and an improvement in FCR of broilers with increasing levels of energy in diets, with and without GAA addition. Solving the equivalence equation, by applying each of the weighted average energy levels studied. indicates the GAA equivalence of 133, 103, 74, and 44 kcal/kg of diet. In conclusion, GAA supplementation improves broilers’ efficiency of energy use; the average ME equivalence of 600 mg/kg of GAA is 88.5 kcal/kg.


Introduction
Guanidinoacetic acid (GAA), the common name of N-(aminoimino-methyl)-glycine, is the precursor to creatine, which, together with phosphocreatine, is inextricably involved in cellular energy metabolism through adenosine triphosphate (ATP) regeneration (Portocarero and Braun, 2021).Guanidinoacetic acid is methylated to creatine by the action of enzyme S-adenosylL-methionine:N-guanidino acetate methyltransferase, which, in poultry, is also expressed in the kidneys as well as in the liver (Van Pilsum et al., 1972).The GAA supplementation to broilers has been found to promote growth performance, enhance breast meat yield, and improve feed conversion ratio (FCR; Oviedo-Rondón and Córdova-Noboa, 2020; Zarghi et al., 2020;de Souza et al., 2021).These effects may be partially related to significant increases in high-phosphate energy metabolites in muscle (DeGroot et al., 2018;Majdeddin et al., 2020).In addition, improvements in energy utilization by birds have been linked to better feed utilization (Khajali et al., 2020).
De novo synthesis of GAA requires amino acids glycine and arginine as precursors.Several studies on broilers have been performed to explore the potential of GAA as a "spare" of arginine (Ale Saheb Fosoul et al., 2019;DeGroot et al., 2019).Other studies have demonstrated that GAA supplementation can improve the energy use efficiency in broilers (Mousavi et al., 2013;Ale Saheb Fosoul et al., 2018).In poultry feed, energy is the most expensive component, accounting for 70% of feed cost (Pirgozliev and Rose, 1999;Noblet et al., 2022).Nevertheless, according to Khajali et al. (2020), the GAA equivalence in metabolizable energy (ME) in diets for broilers needs to be determined.The first studies with reduced energy in diets for broilers showed that GAA supplementation can contribute the equivalent of 47.8 kcal/kg ME (Çenesiz et al., 2020) and 50.0 kcal/kg ME (Ceylan et al. 2021).
In this study, we hypothesized that GAA supplementation may improve the energy use efficiency and, consequently, the performance of broilers.Therefore, we evaluated how GAA addition in diets with various energy contents affects the performance of broiler chickens; we also estimated an equivalence in ME of GAA.

Ethical matters
The Institutional Animal Care and Use Committee approved all animal handling procedures (case number 34/2020), and the experiment was conducted according to the experimental protocol for the use of live birds from the Brazilian College of Animal Experimentation.
Based on their body weight, we assigned a total of 1,280 one-day-old broilers to a completely randomized design with eight treatments, eight repetitions, and twenty birds per experimental unit.The birds were housed in 64 floor pens (2 m 2 ), each equipped with four nipple drinkers and a feed dispenser.
Corn-soybean meal-based diets were formulated to meet the nutritional recommendations given by Rostagno et al. (2017) according to phase, except for ME levels (Table 1).Basal diets contained 2,775, 2,875, and 2,975 kcal/kg in the phases of 1 to 7, 8 to 21, and 22 to 42 days, respectively.Treatments were based on four ME levels per phase (Table 2) and the inclusion of 0 or 600 mg/kg GAA (CreAMINO ® , minimum 96% GAA, AlzChem, Trostberg, Germany).The increases in the ME levels of the basal diets were 75, 150, and 225 kcal/kg based on the experimental treatments.These energy density increases were carried out exclusively with the addition of soybean oil instead of the inert.The addition of GAA to the experimental diets was also in place of the inert.The diets were prepared in mashed form.Birds had free access to water and feed throughout the experimental period (1 to 42 days of age), and were exposed to 24 h of light from ages 1-14 days old, after which an 18 h light:6 h dark cycle was implemented until the end of the experiment.
Metabolizable energy equivalence of guanidinoacetic acid in corn soybean meal-based broiler diets Salgado et al. 3

Performance and carcass characteristics
Birds and feed leftovers, were weighed at 42 days of age to calculate feed intake (FI), weight gain (WG), and FCR.Mortalities were recorded throughout the experimental period, and the necessary corrections of performance data were calculated.At 42 days old, two birds with weights closest to the average weight of their respective experimental unit were selected.After 8 h of fasting, these broilers were euthanized and slaughtered to measure the yield of carcass, breast, and thigh with drumstick, as well as the relative weight of abdominal fat.Carcass yield was calculated in relation to living weight before slaughter (carcass weight × 100/live weight) and breast and thigh with drumstick yield as a function of carcass weight (part weight × 100/ carcass weight).The relative weight of abdominal fat was calculated in relation to the birds' live weight before slaughter.

Statistical analysis and ME equivalence calculations
For each variable, the analysis of variance was performed according to the following general model: in which Y ij is the measured dependent variable, μ is the overall mean, α i is the effect of treatments, and ε ij is the random error.
Analyses were carried out using the PROC GLM of SAS (Statistical Analysis System, version 9.4).The significance of the effects was tested at the 5% probability level.To assess the effect of including GAA at each energy level, contrast analyses were performed.Linear equations for energy levels with or without GAA supplementation were also estimated using the PROC REG of SAS.Significance for each of the regression model parameters was tested at the 5% probability level using Student's t test.
The ME equivalence of the GAA was estimated with a methodology adapted from Jendza et al. (2006) and Stefanello et al. (2017).Linear effects of increasing ME in diets with or without GAA addition were tested.Regression equations of ME levels were generated for FCR, and an equivalence equation was obtained by equating the two linear equations estimated as follows: Y = a + bX 1 (FCR response according to ME levels in diets with GAA) (2) Y = a + bX 2 (FCR response according to ME levels in diets without GAA) Equivalence equation: in which Y is the FCR response; X 1 is the ME level in diets with GAA; X 2 is the ME level in diets without GAA; a is the intercept in each respective equation; and b is the slope in each respective equation.
The equivalence equation was solved by substituting the weighted average energy levels studied in X 1 and obtaining X 2 .The equivalence in ME of the GAA was estimated at each energy level studied by subtracting X 1 from X 2 , and the average of the estimates was calculated.

Results
There was no effect of GAA supplementation on the FI of broilers at any energy level studied (P>0.05;Table 3).However, GAA supplementation increased the WG of broilers at the energy level of 2,908 kcal/kg (P = 0.036) and improved the FCR at the energy levels of 2,908 kcal/kg (P = 0.004) and 2,983 kcal/kg (P = 0.049).Regarding the energy levels, there was a linear reduction in the FI of broilers with increasing levels of energy in the diets without (P = 0.015) and with (P = 0.018) GAA addition (Table 4).The FCR improved linearly with increasing levels of energy in the diets without (P<0.001)and with (P = 0.008) GAA.Solving the equivalence equation (by applying the weighted average energy levels studied) indicates that the ME equivalence of GAA were 133, 103, 74, and 44 kcal/kg of diet, with an average equivalence of 88.5 kcal/kg (Table 5).
The carcass, breast and thighs with drumstick yield, and abdominal fat of the birds were not influenced by GAA supplementation at any energy level studied (P>0.05;Table 6); these parameters were also unaffected by the energy levels in the diets (P>0.05).

Discussion
In this study, we hypothesized that GAA supplementation may improve broilers' energy use efficiency.This was confirmed by the improvement in WG and FCR of broilers fed diets with the two lower energy levels studied.According to the recommendations of the National Research Council (NRC, 1994), along with more recent standards adopted by the Brazilian Poultry Sector (Rostagno et al., 2017), these ME levels are a performance-limiting factor.The improvements in WG and FCR may be explained by higher creatine and phosphocreatine levels and higher ATP:ADP and phosphocreatine:ATP ratios in the   muscles of broilers fed diets with GAA (Yazdi et al., 2017;DeGroot et al., 2018;Majdeddin et al., 2020); these improved parameters indicate more efficient energy metabolism.The phosphocreatine:ATP ratios in the breast muscles of broilers were reported to be 28.4 and 20.3 for those that received GAA at 600 mg/kg and for the control group, respectively (Yazdi et al., 2017).Ale Saheb Fosoul et al. (2018) reported that the enhancement in the buffering capacity for ATP in the muscles exerted by supplemental GAA affects the metabolism of energy in broiler chickens fed diets with energy reduction, resulting in an improved FCR.In addition to functioning directly in muscle accretion as the precursor to creatine, dietary GAA can also effectively "spare" arginine from being used for GAA synthesis, so that the arginine may be used for muscle accretion and other physiological functions (Portocarero and Braun, 2021).As in the present study, Mousavi et al. (2013) reported that GAA supplementation can potentially improve the FCR and energy efficiency of broilers.
Previous study showed that the use of GAA improved the breast meat yield, but without effect on carcass and other cuts (Córdova-Noboa et al., 2018).In the present study, no effects of GAA were observed on carcass, breast, and thighs with drumstick yield.Similar results were observed by Mousavi et al. (2013), who also evaluated the effect of GAA addition to diets containing different levels of ME.
With increasing levels of ME in diets without and with GAA addition, the birds reduced their FI.This result was expected, based on the literature, because broilers may adjust their FI in response to their energy needs (Leeson et al. 1996;Hu et al., 2021).This is linked to metabolic signalization.Hu et al. (2019) reported that the central adenosine monophosphate-activated protein kinase signaling pathway and appetite are modulated in accordance with the energy level in the diet to regulate nutritional status and maintain energy homeostasis in broilers.
With the reduction in FI and no effect on WG, the FCR of broilers improved with increasing levels of energy in the diets with and without GAA addition, in accordance with several reports (Leeson et al., 1996;Ale Saheb Fosoul et al., 2018;Hu et al., 2021).The FCR responses observed in this study suggest an average ME equivalence of 88.5 kcal/kg, different from the values of 47.8 kcal/kg ME (Çenesiz et al., 2020) and 50.0 kcal/kg ME (Ceylan et al., 2021) observed in studies with reduced energy in diets and the same GAA supplementation.However, further research is needed to validate this dietary ME in practical diets.

Table 3 -
Growth performance of broiler chickens from 1 to 42 days of age ,200 kcal/kg, from 1 to 7, 8 to 21, and 22 to 42 days of age, respectively.Means within each column followed by different letters differ by Tukey's test (P<0.05).

Table 4 -
Linear regression equations estimated for each variable on the response of energy levels with or without guanidinoacetic acid (GAA) supplementation FI -feed intake; FCR -feed conversion ratio; SE -standard error.

Table 5 -
Equivalence equation for feed conversion ratio (FCR) and to estimate the energy equivalence of guanidinoacetic acid (GAA)