Ethical considerations

The study was approved by the Ethics Committee on Animal Use of Universidade Federal de Goiás (UFG; protocol nº 011/16).

Location and animals

The experiment was conducted at UFG, Goiás, Brazil. A total of 252 one--day old male chicks (Cobb lineage) were used, distributed in a completely randomized design with 3 treatments, 7 replicates, and 12 chickens per replicate (21 experimental units).

Chickens were placed in five floors of steel battery cages (0.77 x 0.74 x 0.23 m), equipped with drinkers, gutter feeders and metal trays to collect excreta. Batteries were kept inside barns, and ventilation was controlled with curtains. Maximum and minimum temperatures were recorded twice a day by thermometers distributed throughout the barn. Incandescent lamps were used to provide luminosity and heating support. Temperatures recorded during the experiment in the periods from 1 to 7, 8 to 14, and 15 to 21 days were, respectively: 24.4 and 33.7; 21.8 and 32.1; 21.5 and 31.8 ºC.

Feed and water were provided ad libitum during the experimental period. Treatments consisted of a basal diet without GAA, and basal with 0.10 or 0.20% GAA. The animals received the dietary treatment until 7 days of age, and the basal diet without GAA until they were 21 days old. The corn-soybean based feed was formulated to meet the minimum nutrient requirements throughout the experimental period, according to ^{Rostagno et al. (2011)} (Table 1).

Weighing of animals and feed was conducted on days 1, 7, 14, and 21 to calculate performance variables. On day 6, two animals per experimental unit were randomly chosen for blood and intestinal sampling. Chickens were fasted for 2 hours and subsequently dulled in a CO₂ gas chamber.

Blood sampling was performed by femoral vein puncture and 2 mL blood was stored in vacutainer tubes with sodium fluoride as anticoagulant and kept in Styrofoam boxes filled with ice. At the UFG’s Departamento de Patología Clínica, tubes without anticoagulant were kept resting for 2 hours, then centrifuged and the resulting serum was used to determine creatinine and creatinine-kinase levels. Tubes containing anticoagulant went through the same centrifugation process, with plasma collected to determine blood glucose levels. Determinations were carried out using specific commercial kits in an automated biochemical analyser (CM 200, Wiener Lab Group®). Sampled intestines were weighted, and their lengths measured with a measuring tape.

Two metabolism assays were conducted: one from the 3rd to the 7th day and the other from the 17th to the 21st day of age. The nutrient assimilation coefficient in feeds was determined from total excreta, following ^{Sakomura and Rostagno (2007)} methods. Excreta were collected and kept frozen until further analysis. At the end of the sampling period, excreta were thawed, homogenized, and 400 g of sample were dried in a forced-air oven at ±55 ºC for 72 hours. Then, samples were weighed and ground in a Willey mill for analysis. The analysis was carried out in the Laboratório de Nutrição Animal at UFG. Dry matter, crude protein, and ether extract from excreta and feed samples were determined according to the methods described by ^{Silva and Queiroz (2009)}.

Statistical analysis

Data were analysed using the R software. An ANOVA was performed, and averages were compared using the Tukey test. Adopting α=0.05, and considering a tendency when α was between 0.05 and 0.10.

Performance

Supplementing GAA in broiler feed during the pre-initial period from 1 to 7 days did not affect weight gain and food consumption (p>0.05) (Table 2). However, we observed a tendency of improvement for feed conversion (p<0.08) in animals that were fed 0.20% GAA compared to the other treatments.

From 1 to 14 days, animals that received the diet containing 0.20% GAA until 7 days of age showed a better feed conversion at 14 days (p<0.05) compared to animals that received the remaining treatments. The 0.20% GAA broiler chickens also showed a tendency (p<0.07) of a greater weight gain in this same period, which explains the improved feed conversion results. Regarding the use of GAA in experimental feeds at the pre-initial period, no residual effect was verified (p>0.05) on broiler performance until 21 days of age.

Creatinine, creatine kinase (CK), and blood glucose

The concentration of CK in the blood of animals that received 0.20% GAA was significantly higher (p<0.01) when compared to those that received other treatments (Table 3).

Blood creatinine values at six days of age presented no difference (p>0.05) between animals that received treatments with GAA inclusion. Blood glucose concentration also did not show any differences (p>0.05) between treatments.

Nutrient assimilation

The dry matter (DMAC), crude protein (CPAC), and ether extract (EEAC) assimilation coefficients in the periods ranging from 3 to 7 and 17 to 21 days are shown in Table 4. In the period from 3 to 7 days, no difference was observed (p>0.05) forboth DMAC and EEAC. Asignificant difference was found for CPAC (P=0.0306), which was higher when animals received 0.20% GAA but did not differ when compared to the group that received 0.10% GAA. No difference (p>0.05) was found between treatments for the period from 17 to 21 days of age. Providing feeds with GAA inclusion during the pre-initial period did not influence the assimilation coefficient in the period from 17 to 21 days.

Intestine weight and length

No difference was found between treatments for intestine weight and length (p>0.05; Table 5).

Performance

We included GAA in broiler chicken diets to improve performance during the first days of life. GAA produces creatine without consuming essential amino acids in the process, which can then be used by the organism to perform other functions, such as protein synthesis and to increase muscle mass. Thus, 0.20% GAA inclusion showed potential for improvement of poultry performance in the period from 1 to 7 days and had a positive effect on feed conversion from 1 to 14 days.

The first seven days of life are determinant for the optimum development of broilers. Therefore, good results should be pursued during this period, as losses are not recovered with compensatory growth until the end of the cycle. This fact can explain the improvement in feed conversion in animals that received a diet containing GAA during the first 7 days of life, which was reflected in the period from 1 to 14 days. In a study by ^{Bryant-Angeloni (2010)}, the inclusion of GAA in arginine-deficient diets yielded no improvement on weight gain but had the opposite effect on feed conversion. On the other hand, when GAA was added to diets containing adequate arginine concentration, improvement was observed in both weight gain and feed conversion. These results confirm that dietary inclusion of GAA can reduce arginine use, thus improving animal performance. This was also observed by ^{Lemme et al. (2007)}, who included GAA in male broiler diet between 1 and 41 days of age and found improved feed conversion as well as increased creatine, phosphocreatine, and muscle ATP content. Therefore, energy availability likely increased, affecting the metabolic use of nutrients for growth. Thus, efficient stocking makes energy available in the short term to other metabolic pathways, such as protein synthesis. The improvement in feed conversion can be a reflection of these effects.

Inclusion of 0.20% GAA in broiler pre- initial diets proved efficient for improving feed conversion until 14 days of age. Furthermore, performance improvement can be linked to the fact that GAA inclusion saves amino acids for endogenous synthesis of CRE so they can be used for protein synthesis and muscle growth instead. Additionally, GAA inclusion makes CRE levels increase, as GAA is a direct precursor of CRE, and the energy available for metabolic processes and performance improvement consequently increases.

Supplementing diets with GAA brings better results during the final rearing period of broilers. ^{Michiels et al. (2012)} reported that GAA supplementation was beneficial in the final period when growth rates are the highest, showcasing the stage when there is better gain:feed relationship. Thus, satisfactory results can be obtained when GAA is supplemented during the final period, considering that greater muscle growth occurs at that stage and more ATP is needed.

In the present study, no residual effect of performance improvement at 21 days was observed. However, the time between birth and adaptation to the experiment was short, in such a way that broilers did not go through a considerable fasting period. Our results suggest that late adaptation, in which the time for chick arrival to the farm enables a longer fast, the response to GAA could be more favorable, which would help to save gluconeogenic precursors.

Creatinine, creatine kinase (CK) and blood glucose

A higher concentration of CK could represent more intense muscle growth (^{Lorsheitter et al., 2015}). Thus, higher CK at the sixth day of life for broilers receiving 0.20% GAA could explain the tendency for improvement in feed conversion from 1 to 7 days, confirming that higher CK blood concentration can be related to more intense muscle growth in this phase.

Creatinine is a product of creatine (CRE) breakdown from muscles. Consequently, blood creatinine levels are closely related to muscle CRE concentration. Creatinine is filtered in the kidneys and excreted in urine; kidney malfunction compromises creatinine filtration decreasing creatinine excretion and increasing its blood concentration. According to ^{Barbosa et al. (2011)}, CRE is excreted through the kidneys before being converted into creatinine. This metabolite exists at low concentrations in poultry serum, usually lower than the minimum level detected by laboratory tests. Normal creatinine levels in poultry are constant, between 0.1 and 0.4 mg/dL (^{Saukas, 1993}). Thus, the creatinine levels observed in the present study are considered normal.

To determine muscle activity, ^{Lemme et al. (2007)} established CRE, creatinine, and guanidinoacetic acid directly from the breast muscle in broilers that received GAA from 1 to 41 days of age. At 41 days of age, muscle CRE content increased about 14% when GAA supplement was raised up to 0.06% in the diet, suggesting that dietary GAA is a strong source of CRE. Furthermore, the number of high-energy molecules increased with GAA supplementation 1-hour post-mortem, suggesting a more efficient energy stocking due to GAA inclusion, which is available in the short term to metabolic functions such as protein synthesis.

Glucose blood concentration in healthy chickens varies from 200 to 500 mg/dL. Chickens that undergo short fast periods keep normal glucose levels through hepatic gluconeogenesis. In longer periods, however, healthy chickens up to 8 days of age do not reduce glucose use as mammals do (^{Schmidt et al., 2007}). In the present study, blood glucose levels were kept in normal levels for healthy chickens.

Nutrient assimilation

The crude protein assimilation coefficient (CPAC), which increased in animals that received 0.20% GAA in the feed during the period between days 3 and 7, demonstrates an improvement in feed protein usage, reflecting on performance, as can be seen in Table 2. This is noticed when observing feed conversion alone from days 1 to 7, and feed conversion and weight gain from days 1 to 14 immediately afterward. The improvement in CPAC increased amino acid availability, caused by GAA inclusion, which might have favored the use of the amino acids from protein deposition, thus demonstrating higher muscle growth, which is also justified by higher blood concentration of CK. Muscle growth is related to the improved tendency of weight gain and feed conversion observed during the pre-initial period when 0.20% GAA was offered.

Intestine weight and length

According to ^{Viola et al. (2008)}, increase in intestinal mass and length is related to increased villi size and crypt depth. Some ingredients used in broiler diets can have trophic and enterocyte-stimulating effects causing an increase in intestinal mass and improvement of nutrient-absorbing capacity. In the present study, GAA inclusion apparently did not alter intestinal morphology. The GAA did not show activities that stimulate intestinal mass growth during the pre-initial rearing period.

Studying the effects of GAA inclusion is necessary, mostly because GAA methylation to CRE consumes many methyl groups from methionine. According to ^{Michiels et al. (2012)}, dietary GAA supplementation increases the demand for methylation. In methylation reactions, homocysteine is produced (^{Deminice et al., 2009}). The great demand for methylation during CRE production causes an accumulation of homocysteine in the blood, as well as a deficiency of methionine, choline, betaine, folic acid, and vitamin B12, increasing the need of these nutrients in the diet. High plasma concentration of homocysteine can cause damage to cell functioning, besides being related to cardiovascular disease (^{Setoue et al., 2008}).

In conclusion, inclusion of 0.20% GAA in pre- initial diets of broiler chickens could improve weight gain and feed conversion between 1 and 14 days of age.