Validation of the Energy Matrix of Guanidinoacetic Acid for Broiler Chickens: Effects on Performance, Carcass Traits, and Meat Quality

1. Introduction

Broiler chicken feed represents approximately 75% of production costs, a figure that is expected to increase due to the commodity nature of animal feed ingredients, especially soybean oil, a primary raw material in feed production [1,2]. Currently, there are few alternative energy sources to soybean oil available for reducing production costs in poultry farming.

Given these challenges, there is a growing need to explore alternative energy sources in broiler diets to enhance both technical and economic sustainability. One potential avenue of study involves improving the metabolic status of animals to enhance cellular ATP production efficiency. Creatine is a molecule that fits this criterion as it serves as a precursor for muscular energy production and promotes muscle growth [3]. Additionally, it plays a direct role in protein accretion by redirecting amino acids arginine, glycine, and methionine, thereby enhancing ATP availability for myosin [4,5].

The premise is that increasing cellular levels of creatine (since endogenous production alone is insufficient for maximum phosphorylation) could enhance organismal energy potential, thereby allowing for reduced caloric content in diets without compromising batch performance. Chickens have limited capacity for endogenous synthesis of creatine, necessitating supplementation through feed sources. Animal-origin ingredients are rich sources of creatine, although their levels diminish significantly due to thermal processing, leading to variability [6]. Moreover, monohydrate creatine, while an added formulation, is considered unstable during feed manufacturing processes [7].

The inclusion of guanidinoacetic acid (AGA) in poultry feed has potential as a cellular creatine precursor, offering an alternative to reduce dietary energy content (by decreasing soybean oil levels), and showing strong potential for use in poultry farming. AGA serves as a creatine precursor in the liver and operates within the avian metabolic biochemical framework, where creatine acts as a phosphorus transporter in the mitochondrial electron transport chain’s final step of oxidative phosphorylation, responsible for producing adenosine triphosphate (ATP) molecules for cellular energy [8]. AGA is stable under various conditions, making it a suitable supplement for feed inclusion [9]. The thermoinstability of creatine complicates its inclusion in diets undergoing thermal processing such as pelleting and extrusion. Guanidinoacetic acid is more stable and can withstand these high-temperature physical processes. This situation increases its utility in the poultry industry, as most feed manufacturers employ one of these processes [10]. Studies demonstrate that AGA (0.6–1.2 g/kg feed) is safe, improving performance without compromising health [11]. However, AGA levels > 1.5 g/kg may reduce feed intake and cause renal risks [6,8].

Creatine naturally occurs in animal-derived meals routinely used in poultry diets, leading to scientific debate regarding the effectiveness of adding pure creatine (or its precursors) to feeds containing animal-derived meals. Given its absence in plants, there is a premise that including creatine in diets composed exclusively of plant-derived ingredients may yield better gains compared to diets containing animal-derived ingredients. However, these hypotheses require further substantiation through additional research [8,11,12].

Therefore, this study aims to evaluate whether the addition of guanidinoacetic acid to broiler diets with varying energy levels affects productive performance parameters, hepatic biochemistry, and serum biochemistry.

2. Materials and Methods

2.1. Animals, Housing, Diets, and Experimental Design

The research was conducted at the poultry facilities of UNOESC Xanxerê, using 480 male COBB lineage chickens distributed on the first day of age. The study was approved by the Ethics Committee on Animal Use (CEUA/UNOESC) under approval number 28/2021. It employed a completely randomized experimental design with three treatments (Table 1), each consisting of eight replications with 20 animals per replication. Guanidinoacetic acid was added to the experimental diets (Tables 2–4) at a rate of 600 mg/kg, an amount recommended to contribute 75 kcal/kg to the feed. Diets were provided in mash form and included ingredients of animal origin, such as meat and bone meal.

Table 1. Treatments used.

Table 2. Dietary composition and nutritional values of experimental diets for initial phase (1–21 days) of nutrition programs with different energy levels.

Table 3. Dietary composition and nutritional values of experimental diets for growth phase (22–33 days) of nutrition programs with different energy levels.

Table 4. Dietary composition and nutritional values of experimental diets for final phase (34–42 days) of nutrition programs with different energy levels.

The animals were obtained from a commercial hatchery (GEAL Hatchery, Xanxerê, SC, Brazil) at one day of age, after being vaccinated against Marek’s disease post-hatching. They were then transferred directly to the experimental aviary, where they were raised following commercial farm standards and breed manual guidelines. They were housed in 2 m² pens with wood shavings bedding, equipped with tube feeders and nipple drinkers, providing ad libitum access to feed and water throughout the experimental period.

2.2. Performance, Carcass, and Organ Yield

Chickens were weighed at 7, 21, and 42 days along with feed leftovers to determine weight gain, feed intake, and feed conversion ratio. At 42 days of age, three birds per experimental unit were euthanized to evaluate carcass yield, specific cuts (wing, drumstick, thigh, back, breast, and abdominal fat), and organ weights (heart, liver, proventriculus, gizzard, small intestine, spleen, and abdominal fat), following animal welfare and euthanasia norms outlined in Resolution n37/2018 [14]. The following calculations were used:

Carcass yield (%) = (carcass weight × 100)/(body weight at slaughter) × 100

Relative weight of cut (%) = (cut weight × 100)/(body weight at slaughter) × 100

Relative weight of organ (%) = (organ weight × 100)/(body weight at slaughter) × 100

2.3. Serum Biochemistry

For biochemical parameter assessment, blood samples (1 mL per animal) were collected via the brachial vein at 42 days of age. Serum was separated by centrifugation and stored at −20 ◦C for subsequent colorimetric enzymatic analysis of glucose (mg/dL), cholesterol (mg/dL), triglycerides (mg/dL), uric acid (mg/dL), total proteins (g/dL), and creatinine (mg/dL) concentrations using commercial kits (Gold Analisa®, Belo Horizonte, MG, Brazil) on a semi-automatic analyzer (Bioplus®, BIO-2000, Barueri, SP, Brazil).

2.4. Bromatological Composition and Lipid Peroxidation Analysis of Breast Meat

Approximately 35 g of chicken breast meat was weighed, frozen, and subsequently lyophilized for dehydration. The dried samples were ground using a laboratory multipurpose mill. The samples were then dried in an oven at 105 ◦C for 8 h to determine total dry matter. Subsequently, samples were incinerated in a muffle furnace at 600 ◦C for 4 h [15]. Nitrogen content was determined by the Kjeldahl method (Method 984.13, AOAC, 1997 [16]) and converted to crude protein (CP) using a correction factor of 6.25. Fat content was determined using the Bligh and Dyer method (1959) [17], which involves fat extraction from samples with chloroform.

Lipid oxidation analysis of breast meat was conducted using the TBA (2-thiobarbituric acid) method, which quantifies malondialdehyde (MDA), a major decomposition product of polyunsaturated fatty acid hydroperoxides formed during oxidation [18]. Results were expressed as milligrams of MDA per gram of sample (mg of MDA/g) based on a standard curve. The methodology followed Pikul et al. [19], with readings on a spectrophotometer (Biospectro® SP-22, Curitiba, PR, Brazil) at a wavelength of 538 nm.

2.5. Statistical Analysis

Experimental results were subjected to Shapiro–Wilk normality tests, and as all data were found to be normally distributed, analysis of variance (ANOVA) was performed. Significant differences among means were determined by Tukey’s test at a significance level of 0.05 using R® statistical software (Posital®, RStudio version 2021.09.0, Boston, MA, USA).

3. Results

3.1. Performance

No significant differences (p > 0.05) were observed in weight, weight gain, and feed intake of the chickens during the 1- to 21-day phase. However, there was a significant reduction (p < 0.001) in feed conversion ratio for chickens receiving diets from the positive control and negative control + AGA groups compared to those in the negative control group (Table 5).

Table 5. Performance of broiler chickens from 1 to 21 days and 1 to 42 days of age, supplemented or not with guanidinoacetic acid, subjected to different metabolizable energy programs in diet.

During the 1- to 42-day period (Table 5), chickens fed diets from the positive control and negative control + AGA groups showed higher body weight, weight gain, and feed intake (p < 0.001). Feed conversion ratio was higher (p < 0.001) in chickens from the negative control group compared to those from the positive control group.

3.2. Organ Yield

No significant effects (p > 0.05) were observed on relative weights of the heart, gizzard, intestines, proventriculus, and spleen in broiler chickens subjected to different treatments. However, there was a difference in relative liver weight (p = 0.037), with chickens from the positive control group having lower liver weights compared to those from the negative control group (Table 6).

Table 6. Relative organ weight of broiler chickens subjected to different dietary energy levels, with or without guanidinoacetic acid supplementation.

3.3. Carcass and Cut Yield

No effects (p > 0.05) were observed on carcass yield, breast, thigh, drumstick, back + wings, and abdominal fat yields in chickens subjected to different treatments in both experiments (Table 7). There was a trend (p = 0.088) towards a difference in breast yield observed in chickens.

Table 7. Carcass and cut yields of broiler chickens subjected to different dietary energy levels, with or without guanidinoacetic acid supplementation.

3.4. Serum Biochemistry

Regarding biochemical data (Table 8), chickens receiving guanidinoacetic acid supplementation in their diet showed higher glucose levels (p < 0.001), lower cholesterol levels (p = 0.013), lower triglyceride levels (p < 0.005), and lower total protein levels (p < 0.001) compared to the negative control group. Creatinine levels (p = 0.056) were also higher in control treatments, with no changes observed in uric acid analysis (p = 0.061).

Table 8. Serum biochemical analysis of broiler chickens subjected to different dietary energy levels, with or without guanidinoacetic acid supplementation.

3.5. Bromatological Composition and Lipid Peroxidation

In the bromatological composition and lipid peroxidation (mg of MDA/g) of the breast muscle in broiler chickens, an increase in crude protein (% in DM) was observed in animals belonging to the NC + AGA treatment group (p < 0.001) compared to those in the negative control group. There was a difference in fat content (%) (p = 0.032), with the negative control group showing a decrease in fat percentage compared to the positive control group. Regarding lipid peroxidation TBARS (p = 0.027), the breast meat of animals in the NC + AGA group showed higher oxidation compared to those in the negative control group (Table 9).

Table 9. Proximate composition and lipid peroxidation of breast muscle of broiler chickens subjected to different dietary energy levels, with or without guanidinoacetic acid supplementation.

4. Discussion

Muscular energy supply is crucial in rapidly growing broiler chickens to achieve their maximum production potential, and energy status is a key determinant of carcass growth. Muscle energy is derived from intracellular ATP, produced through biochemical reactions in glycolysis, the Krebs cycle, and the respiratory chain in mitochondria. Creatine plays a significant role by “recycling” phosphorus molecules within the intracellular environment to facilitate the production of new ATP molecules, bypassing the need for subsequent metabolic reactions [20,21]. Creatine binds with phosphorus to form phosphocreatine, which is then acted upon by the enzyme creatine kinase to regenerate ATP from ADP in mitochondria [20,21]. The interaction between creatine and phosphocreatine with ATP and ADP, respectively, suggests that creatine-loaded muscles have the capacity to enhance growth or work efficiency [12].

Dietary guanidinoacetic acid increases muscle creatine concentrations, leading to improved energy metabolism in the respective tissue [22]. Guanidinoacetic acid serves as a precursor to creatine in the liver, which is then directed to tissues with high energy demands such as skeletal muscle, cardiac muscle, and the brain following its synthesis. In our study, we observed improvements in livestock performance, particularly in feed conversion efficiency, through the addition of guanidinoacetic acid to diets. Supplementation with AGA can reduce serum creatinine levels, due to greater efficiency in the use of phosphocreatine for ATP synthesis and arginine sparing, which reduces the endogenous production of creatine and its residual metabolite (creatinine) [23,24]. These findings align with those reported by Khajali et al. [25], who also found improved feed conversion efficiency in poultry supplemented with guanidinoacetic acid. This improvement occurred without changes in feed intake, indicating increased energy efficiency in chickens with guanidinoacetic acid supplementation [26].

There has been a scientific controversy regarding the addition of guanidinoacetic acid, or even creatine, to diets already containing animal-derived ingredients, as these typically contain creatine naturally derived from muscle tissues. However, the processing of these ingredients in rendering plants involves high-temperature digestion, which destroys creatine due to its thermolabile nature, thereby supporting the beneficial effects of exogenous supplementation on performance in poultry. Lemme et al. [22] found that supplementation with guanidinoacetic acid improves animal performance in diets containing fish meal, consistent with findings by Córdova-Noboa et al. [27], who demonstrated improved feed conversion and weight gain in animals supplemented with guanidinoacetic acid in diets containing animal meals. Additionally, Esser et al. [28] reported that feed conversion was better in animals supplemented with guanidinoacetic acid in diets containing animal meals compared to other treatments tested. Thus, guanidinoacetic acid supplementation at different stages of animal growth can mitigate the adverse effects of energy reduction in poultry diets, compared to the group without supplementation [29].

Additionally, there is another physiological mechanism that helps elucidate the action mechanism of the molecule in this study. Studies report that the improved performance observed in guanidinoacetic acid-supplemented broiler chickens may be attributed to its ability to spare arginine and glycine in metabolism [6,14,24,30], as the body produces guanidinoacetic acid in the liver using arginine and glycine as precursors. Supplementing this compound allows the organism to spare and redirect these amino acids for other functions, such as protein synthesis, resulting in improved animal performance [31]. This arginine-sparing function is practically significant in the nutrition of broiler chickens, as they lack a functional urea cycle and are entirely dependent on dietary arginine [25].

Dietary arginine is required for the synthesis of compounds such as ornithine, proline, citrulline, glutamate, and for protein synthesis. It also increases the release of insulin, growth hormone, and IGF-I into the bloodstream, playing roles in both catabolic and anabolic events in skeletal muscle, adding to myofibrillar protein, which is crucial for the process of muscle hypertrophy [32,33]. In our results, particularly observed in Experiment I, the p value of 0.088 for breast yield (Table 8) approached statistical significance, indicating better utilization of dietary amino acids. Studies by Fernandes et al. [34] and CórdovaNoboa et al. [27] have described improvements in breast yield in chickens supplemented with guanidinoacetic acid. A study by EFSA [9] demonstrated that guanidinoacetic acid supplementation at doses of 800 mg/kg in the diet increased breast weight and reduced abdominal fat in animals. According to Wyss and Kaddurah-Daouk [20], supplementation of diets with creatine, even when used correctly, may not increase muscle mass due to variability in individual absorption, transport, and intramuscular storage.

There were no observed changes in the relative organ weights in most cases, except for differences in liver weight between positive control and negative control. Since guanidinoacetic acid does not exert digestive effects, it is expected not to affect these organs. Liver lipid metabolism is heavily burdened in poultry, where there is significant fat mobilization to this organ in certain situations to catabolize fatty acids [35].

It was observed that the addition of guanidinoacetic acid to the diet improves glucose availability and consequently energy, as it is used as an energy source by the organism. It is noted that in the experiment, with energy levels recommended by Rostagno et al. [13], there was a decrease in cholesterol and triglycerides in animals supplemented with guanidinoacetic acid, related to the reduction in vegetable oil. Conversely, animals fed a high-energy diet showed increased levels of cholesterol and triglycerides, as vegetable oil is an unsaturated fatty acid that is easier to digest and absorb. This set of information allows us to infer that the organism (especially muscle tissue) used intracellular energy sources more efficiently, saving glucose at a general metabolic level.

Following the same logic, serum triglyceride levels decreased due to less fat mobilization needed to meet the organism’s energy demand. Cholesterol, on the other hand, was reduced due to its lower demand as a lipid transporter in the organism, reflecting reduced overall lipid mobilization. A reduction in total protein levels was also observed with the decrease in dietary energy levels, which can be explained by the reduced need for lipoprotein transporters in the blood, as the lower presence of lipids in the diet reduced the requirement.

Serum biochemical analysis reflects the metabolic status of the animals, enabling the assessment of tissue damage, organ function issues, and the adaptation of animals to physiological and nutritional challenges [36]. The biochemical profile allows us to evaluate whether the use of additives or some exogenous molecules can be safely conducted in the animal organism. In our study, there was a reduction in almost all evaluated parameters, except for uric acid levels, which remained constant. This situation allows us to infer that there are no metabolic risks associated with the use of guanidinoacetic acid, indicating its safe use as a molecule.

Since guanidinoacetic acid is considered a saver of dietary arginine, it has been studied as an alternative for modulating lipid deposition and promoting protein synthesis. Thus, the use of a lipid source and supplementation with guanidinoacetic acid may explain the higher protein content and lower percentage of fat in chicken breast meat found in this study. In literature, several studies have found an increase in breast yield [25,29]. Increased breast yield is one of the most sought-after parameters in poultry farming recently, as it is one of the most financially representative cuts of the carcass, contributing to the technical and economic viability of using the additive in question.

Excessive production of reactive oxygen species (ROS) is detrimental to normal metabolism and can cause cellular damage through lipid peroxidation and protein oxidation [37,38]. Peroxidation occurs as a result of oxidative attack on membrane phospholipids [39]. Lipid peroxidation primarily affects cell membranes, altering their structure and permeability. This leads to the loss of selective ion exchange and the leakage of organelle contents, generating cytotoxic products such as malondialdehyde (MDA), ultimately resulting in cell death [40,41]. The increased formation of free radicals may result from elevated oxygen consumption and the activation of specific metabolic pathways related to muscle growth.

According to Wang et al. [42], the addition of guanidinoacetic acid improves antioxidant status by increasing total antioxidant capacity and the activities of several antioxidant enzymes. Metabolites related to guanidinoacetic acid (creatine and arginine) may be capable of scavenging free radicals, suggesting an indirect antioxidant effect of its use. Creatine, the final product of guanidinoacetic acid utilization, is believed to possess antioxidant capacity in some studies [43,44], but is reported to reduce antioxidant status in others [45], which is consistent with the findings of our study. The increase in MDA observed in the study may reflect transient oxidative stress due to the greater muscular energy demand induced by AGA, exacerbated by the reduction of dietary antioxidants (such as vitamin E) in the low-energy diet. However, further information is needed to understand how guanidinoacetic acid affects the antioxidant system [39].

5. Conclusions

The addition of guanidinoacetic acid can replace vegetable oil as an energy source for broiler chickens, ensuring maintenance of livestock performance and maintaining weight development parameters of organs, carcass, and carcass cuts of the birds.

This article was originally published in Poultry 2025, 4, 30. https://doi.org/ 10.3390/poultry4030030. This is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).