Animal Feed Science and Technology 315 (2024) 116010

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Animal Feed Science and Technology

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Effect of dietary non-essential amino acid sources on performance,

nitrogen utilization and blood parameters for broiler chickens fed
a low-protein diet
Rosana Cardoso Maia , Rafael de Sousa Ferreira *, Luiz Fernando Teixeira Albino ,
Arele Arlindo Calderano , Bruno Damaceno Faria , Diego Ladeira da Silva,
Thaisa Pereira de França, Horácio Santiago Rostagno
Universidade Federal de Viçosa, Departamento de Zootecnia, Viçosa, MG, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Two experiments were carried out to evaluate sources of non-essential amino acids (NEAA) in
amino acid low-protein diets and their effects on performance, nitrogen retention, and blood parameters in
essential-to-total nitrogen ratio broiler chickens. In the first experiment: three dietary treatments were formulated with 10
nutrition
replicate pens and five male Cobb-500 broiler chickens per experimental unit: T1, positive control
poultry
protein
(PC) diet with 222.5 g/kg crude protein (CP) and an essential-to-total nitrogen ratio (eN-to-tN) of
0.4766; T2, negative control (NC) with 205 g/kg CP and an eN-to-tN of 0.5156; T3, 205 g/kg CP
+ 2.8 g/kg of glutamate (Glu), and an eN-to-tN of 0.5163. In the second experiment: six dietary
treatments were fed to 10 replicate pens of five male Cobb-500® broiler chickens as follows: T1,
PC with 222.5 g/kg CP and an eN-to-tN of 0.4706; T2, NC diet with 205 g/kg CP and an eN-to-tN
of 0.5695; T3, NC + 3.846 g/kg glycine (Gly) and an eN-to-tN of 0.4706; T4, NC + 4.041 g/kg
alanine (Ala) and an eN-to-tN of 0.4706; T5, NC + 7.287 g/kg Glu and an eN-to-tN of 0.4706; T6,
NC + 4.080 g/kg NEAA mixture (Gly + Ala + Glu) and an eN-to-tN of 0.4706. Performance was
measured after 21 days. Excreta were collected to calculate nitrogen utilization from day 14–21.
Blood was collected to quantify uric acid, total protein, albumin and globulin and to evaluate
nitrogen utilization. When crude protein was reduced from 222.5 to 205 g/kg CP, it resulted in a
worsening of body weight gain (BWG) and feed conversion ratio (FCR). A low CP diet supple­
mented with glutamate increased BWG and decreased the FCR. The values of nitrogen intake (NI)
and nitrogen balance (NB) were higher (P<0.05) in the PC and NC + Glu treatments than in the
NC. In the second experiment, NC + Gly resulted in lower BWG and higher feed intake (FI), but
the FCR was better than in the other treatments. Serum uric acid was highest in the NC + Glu, Ala
and NEAA mixture. The NI was lowest in the NC + Gly treatment, whereas in this same treatment
we observed the highest NB. A low CP diet supplemented with glutamate, alanine and NEAA and

Abbreviations: AA, amino acid; Ala, alanine; Albu, albumin; BWG, body weight gain; CP, crude protein; DEB, dietary electrolyte balance; EN-to-
tN, essential to total nitrogen; EAA, essential amino acid; FCR, feed conversion ratio; FI, feed intake; Glu, glutamine; Gly, glycine; Glob, globulin; N,
nitrogen; NB, nitrogen balance; NC, negative control; NE, nitrogen excreted; NEAA, non-essential amino acid; NI, nitrogen intake; NUE, nitrogen
utilization efficiency; NUEG, nitrogen utilization efficiency for gain; PC, positive control; SUA, serum uric acid; SUANg, serum uric acid per g of NI;
TP, total protein.
* Correspondence to: Rua Gomes Barbosa, 102, Centro, Viçosa, Minas Gerais 36570-101, Brazil.
E-mail addresses: faelherreira@[Link], [Link]@[Link] (R. de Sousa Ferreira).

[Link]
Received 30 November 2022; Received in revised form 11 March 2024; Accepted 24 May 2024
Available online 28 May 2024
0377-8401/© 2024 Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

an eN:tN of 0.4706 improved the broilers’ BWG, FCR, and nitrogen utilization efficiency more
than a low CP diet with 205 g/kg CP and an eN:tN ratio of 0.5695.

1. Introduction

Low crude protein (CP) diets for broiler chickens are related to lower feed costs and lower environmental impact due to reduced
nitrogen excretion and ammonia emissions (Belloir et al., 2017; Greenhalgh et al., 2020; Cappelaere et al., 2021). Furthermore, there
are lower incidences of footpad dermatitis and breast blisters due to the drier litter associated with low CP diets (Swiatkiewicz et al.,
2017). However, the reduction in crude protein can have a negative effect on the productive efficiency of broiler chickens (Belloir
et al., 2017; Kidd et al., 2021). This is probably due to the limitation on the concentration of essential amino acids (EAA), which are
considered indispensable in the animal diet (Chrystal et al., 2020). However, positive economic, environmental and performance
improvements can be achieved through the use of “the ideal protein concept”, whereby diets with lower amounts of crude protein can
present an ideal balance of EAA for birds through the dietary addition of crystalline amino acids (Wu et al., 2014).
Although the use of the ideal protein concept in broiler nutrition can improve animal performance when compared to a low CP diet
without EAA supplementation (Wu et al., 2014; Kidd et al., 2021), this approach has been questioned. This is because when we reduce
the level of protein, we also reduce the non-specific nitrogen that could be used for the synthesis of non-essential amino acids (NEAA)
(Maia et al., 2021). In high CP diets, for example, NEAA can be synthesized from excess EAA as EAA provide non-specific nitrogen for
their synthesis. However, in a low CP diet this is not possible, because if NEAA synthesis from EAA occurs, NEAA become limiting for

Table 1
Ingredients and nutrient composition analysis of the basal diet (g/kg) in the first experiment.
Treatments PC NC NC + Glu

CP (g/kg) 222.5 205 205

eN:tN ratio 0.4766 0.5148 0.4766 (+Glu)
Ingredients
Corn 455.80 516.30 516.30
Soybean meal 372.80 315.00 315.00
Corn gluten meal 20.00 20.00 20.00
Soybean oil 34.00 23.70 23.70
Dicalcium phosphate 15.16 15.65 15.65
Limentone 8.84 8.83 8.83
Salt 4.00 2.00 2.00
Sodium bicarbonate 1.27 4.21 4.21
L-Lysine HCl (790 g/kg) 1.41 3.15 3.15
DL-Methionine (990 g/kg) 2.52 3.00 3.00
L-Threonine (980 g/kg) 0.17 0.94 0.94
L-Valine (965 g/kg) - 0.60 0.60
L-Arginine (985 g/kg) - 0.57 0.57
L-Isoleucine (990 g/kg) - 0.11 0.11
Premixa 3.95 3.95 3.95
Acid-insoluble ash 10.00 10.00 10.00
L-Glutamate - - 28.00
Corn starch 70.00 70.00 42.00
Chemical composition
Metabolizable energy, kcal/kg 3,075 3,075 3,075
Crude protein, analyzed g/kg 219.0 204.3 224.9
Calcium, g/kg 8.19 8.19 8.19
Available phosphorus, g/kg 3.91 3.91 3.91
Sodium, g/kg 2.10 2.10 2.10
Dig Lysine, g/kg 11.74 11.74 11.74
Dig Met + Cys, g/kg 8.46 8.46 8.46
Dig Threonine, g/kg 7.63 7.63 7.63
Dig Tryptophan, g/kg 2.45 2.45 2.45
Dig Arginine, g/kg 13.74 13.74 13.74
Dig Valine, g/kg 9.39 9.39 9.39
Dig Isoleucine, g/kg 8.73 8.73 8.73
Dig Gly + Ser, g/kg 18.38 18.38 18.38
Dig Histidine, g/kg 5.37 5.37 5.37
pH 7.42 6.79 6.61

Crude protein in the diets was analyzed by the Kjeldahl method (AOAC International, 2006).
a
Premix (amount per kg diet): manganese - 77.0 mg, iron - 55.0 mg, zinc - 71.5 mg, copper - 11.0 mg, iodine - 1.10 mg, and excipient q.s. - 1,000 g;
vitamin premix (amount per kg diet): vitamin A - 8250 IU, vitamin D3 - 2090 IU, vitamin E - 31.0 IU, vitamin B1 - 2.20 mg, vitamin B6 - 3.08 mg,
pantothenic acid - 11.0 mg, biotin - 0.077 mg, vitamin K3 - 1.65 mg, folic acid - 0.77 mg, nicotinic acid - 33.0 mg, vitamin B12 - 0.013 mg, selenium -
0.33 mg, and excipient q.s. - 1,000 g; 1 g choline chloride 60%/kg diet; 0.1 g butylated hydroxytoluene/kg diet; 0.55 g sodium salinomycin 12 m/kg
diet; 0.1 g avilamycin 10 mg/kg diet.

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R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

the birds, affecting their performance (Aftab et al., 2006). Therefore, based on these observations, Wu and Li (2022) redefined the ideal
protein concept as being a “correct balance between the EAA and NEAA". They mention that not all NEAA are sufficiently synthesized
by birds and that their supplementation in the diet is necessary to improve broiler performance and health.
Glycine (Gly), alanine (Ala), aspartate (Asp), asparagine (Asx), glutamate (Glu), glutamine (Gln) and proline (Pro) are known as
NEAA. Some researchers have shown the effect of NEAA supplementation in a low CP diet. Awad et al. (2015) observed that a mixture
of NEAA (Gly, Glu, Pro, Ala and Asp) supplementation in low CP diets can improve the performance of broilers fed low CP diets, when
compared to a low CP diet without NEAA supplementation. Dean et al. (2006) reported that the individual supplementation of Glu,
Pro, Ala, and ASP in a low CP diet (160 g/kg CP) did not affect the feed conversion ratio (FCR) of broiler chickens from 1 to 18 d of age.
However, the supplementation of Gly in a low CP diet improved the FCR to a comparable level found in birds receiving a higher CP diet
(220 g/kg CP).
In this context, we hypothesized that the reduction of protein levels associated with the supplementation of EAA and NEAA im­
proves the performance of broilers, in addition to resulting in an improvement in nitrogen retention and blood parameters. Therefore,
two experiments were carried out to evaluate sources of NEAA in low protein diets and their effects on performance, nitrogen

Table 2
Ingredients and nutrient composition analysis of the basal diet (g/kg) in the second experiment.
Treatments PC NC NC + Gly NC + Ala NC + Glu NC + Mix

CP (g/kg) 222.5 185 185 185 185 185

eN:tN ratio 0.4706 0.5695 0.4706 0.4706 0.4706 0.4706
Ingredients
Corn 426.30 489.00 489.00 489.00 489.00 489.00
Soybean meal 384.30 250.40 250.40 250.40 250.40 250.40
Corn gluten meal 20.00 20.00 20.00 20.00 20.00 20.00
Soybean oil 42.60 19.56 19.56 19.56 19.56 19.56
Dicalcium phosphate 15.12 16.51 16.51 16.51 16.51 16.51
Limentone 8.81 8.71 8.71 8.71 8.71 8.71
Salt 4.20 1.51 1.51 1.51 1.51 1.51
Sodium bicarbonate 0.100 5.00 5.00 5.00 5.00 5.00
Potassium carbonate - 3.00 3.00 3.00 5.40 3.00
L-Lysine HCl (790 g/kg) 1.11 5.32 5.32 5.32 5.32 5.32
DL-Methionine (990 g/kg) 2.47 3.83 3.83 3.83 3.83 3.83
L-Threonine (980 g/kg) 0.06 2.05 2.05 2.05 2.05 2.05
L-Valine (965 g/kg) - 2.03 2.03 2.03 2.03 2.03
L-Arginine (985 g/kg) - 2.74 2.74 2.74 2.74 2.74
L-Isoleucine (990 g/kg) - 1.42 1.42 1.42 1.42 1.42
L-Histidine HCl (740 g/kg) - 0.38 0.38 0.38 0.38 0.38
L-Tryptophan (990 g/kg) - 0.25 0.25 0.25 0.25 0.25
Premixa 3.95 3.95 3.95 3.95 3.95 3.95
Acid-insoluble ash 10.00 10.00 10.00 10.00 10.00 10.00
L-Glycine - - 38.46 - - -
L-Alanine - - - 40.41 - -
L-Glutamate - - - - 72.87 -
NEAA mixture (154.5 g/kg N)b - - - - - 40.80
Corn starch 80.00 149.41 114.55 109.00 76.54 108.61
Chemical Composition
Metabolizable energy, kcal/kg 3,075 3,075 3,075 3,075 3,075 3,075
Crude protein, analyzed g/kg 210.9 189.9 227.0 214.0 213.0 219.0
Calcium, g/kg 8.19 8.19 8.19 8.19 8.19 8.19
Available phosphorus, g/kg 3.91 3.91 3.91 3.91 3.91 3.91
Sodium, g/kg 2.10 2.10 2.10 2.10 2.10 2.10
Dig Lysine, g/kg 11.74 11.74 11.74 11.74 11.74 11.74
Dig Met + Cys, g/kg 8.46 8.46 8.46 8.46 8.46 8.46
Dig Threonine, g/kg 7.63 7.63 7.63 7.63 7.63 7.63
Dig Tryptophan, g/kg 2.50 2.50 2.50 2.50 2.50 2.50
Dig Arginine, g/kg 14.01 14.01 14.01 14.01 14.01 14.01
Dig Valine, g/kg 9.51 9.51 9.51 9.51 9.51 9.51
Dig Isoleucine, g/kg 8.88 8.88 8.88 8.88 8.88 8.88
Dig Gly + Ser, g/kg 18.66 18.66 18.66 18.66 18.66 18.66
Dig Histidine, g/kg 5.43 5.43 5.43 5.43 5.43 5.43
pH 6.65 7.50 7.74 7.80 5.58 6.89

CP: crude protein; eN:tN: essential nitrogen:total nitrogen; PC: positive control; NC: negative control; Gly, glycine; Ala, alanine; Glu: glutamate.
Crude protein in the diets was analyzed by the Kjeldahl method (AOAC International, 2006).
a
Premix (amount per kg diet): manganese - 77.0 mg, iron - 55.0 mg, zinc - 71.5 mg, copper - 11.0 mg, iodine - 1.10 mg, and excipient q.s. - 1,000 g;
vitamin premix (amount per kg diet): vitamin A - 8250 IU, vitamin D3 - 2090 IU, vitamin E - 31.0 IU, vitamin B1 - 2.20 mg, vitamin B6 - 3.08 mg,
pantothenic acid - 11.0 mg, biotin - 0.077 mg, vitamin K3 - 1.65 mg, folic acid - 0.77 mg, nicotinic acid - 33.0 mg, vitamin B12 - 0.013 mg, selenium -
0.33 mg, and excipient q.s. - 1,000 g; 1 g choline chloride 60%/kg diet; 0.1 g butylated hydroxytoluene/kg diet; 0.55 g sodium salinomycin 12 mg/kg
diet; 0.1 g avilamycin 10 mg/kg diet.
b
Nonessential amino acids mixture: 50% L-Glycine, 30% Alanine, 20% Glutamate

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R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

retention, and blood parameters in broiler chickens.

2. Materials and methods

2.1. Ethical matters

The Animal Care and Use Committee of the Universidade Federal de Viçosa, Brazil, approved all animal handling procedures
(protocol number 106/2014), and the experiment was conducted according to the experimental protocol for the use of live birds of the
Brazilian College of Animal Experimentation (Marques et al., 2009).

2.2. Birds, experimental design, and diets

Two experiments were carried out on the poultry farm at the Universidade Federal de Viçosa located in the state of Minas Gerais,
Brazil. Male Cobb-500® broiler chickens were used during the starter phase (8–21 days of age). The chickens were raised until 8 days
of age in a masonry house divided into protected circular pens containing a litter of wood shavings, tube feeders and manual drinkers
with ad libitum access to feed and water. The lighting program consisted of 24 h of light, and temperature was maintained at 32ºC in
the first week and gradually reduced according to recommendations in the Cobb® guidelines. A corn and soybean meal pre-starter diet
was formulated to meet the nutrient recommendations proposed by Rostagno et al. (2011). At 8 days, the birds were weighed and
allocated in a completely randomized design to the dietary treatments and placed in wire-floored cages (68 cm×37 cm) (500 cm2/bird)
in a four-level battery equipped with a trough feeder and a nipple drinker.
In the first experiment, three dietary treatments were formulated with 10 replicate pens and five birds per experimental unit,
totaling 150 birds. The first treatment was a positive control (PC) diet with 222.5 g/kg CP and an essential-to-total nitrogen ratio (eN-
to-tN) of 0.4766. The second treatment was a negative control (NC) diet with 205 g/kg of CP and an eN-to-tN ratio of 0.5148. The third
treatment was the negative control supplemented with glutamic acid to meet the same eN-to-tN ratio as the positive control of 0.4766
(Table 1). In the second experiment, 300 male broilers were used and divided into six dietary treatments, with 10 replicate pens of five
birds each. Treatments consisted of six eN-to-tN ratios: T1 = positive control (PC) diet with 222.5 g/kg CP (eN-to-tN ratio 0.4706); T2
= negative control (NC) diet with 185 g/kg CP (eN-to-tN of 0.5695); T3 = NC + Glycine (eN-to-tN of 0.4706); T4 = NC + Alanine (eN-
to-tN of 0.4706); T5 = NC + Glutamic Acid (eN-to-tN of 0.4706); T6 = NC + Mixture (Gly+Ala+Glu) (eN-to-tN of 0.4706) (Table 2).
The experimental diets were formulated by the addition to the basal feed of an appropriate amount of the crystalline amino acids, that
is L-Lysine HCL, L-Threonine, L-Valine, L-Arginine HCL, L-Isoleucine, L-Histidine, L-Tryptophan (Ajinomoto Inc., Tokyo, Japan; 780 g
lysine/kg, 980 g threonine/kg, 965 g valine/kg, 985 g arginine/kg, 990 g isoleucine/kg, 740 g histidine/kg, and 990 g tryptophan/kg),
and DL-Methionine (EVONIK, Germany; 990 g methionine/kg). Treatments were formulated by supplementing the basal diet with
NEAA (L-Glycine, L-Alanine, and L-Glutamate; Ajinomoto Inc., Tokio, Japan) as a replacement for corn starch.
The nitrogen ratio was calculated for all diets considering the requirement of essential amino acids multiplied by their nitrogen
content. Total nitrogen was calculated as the sum of the nitrogen content for all dietary components. All diets were supplemented with
10 g/kg of celite (acid-insoluble ash - AIA) (Imerys, Arica, Chile). Potassium carbonate and sodium bicarbonate were used to maintain
the same dietary electrolyte balance (DEB, approximately 200 mEq/kg) and pH in the diets.

2.3. Performance, sample collection, and calculations

During the experimental period (8th to 21st day), broiler performance was evaluated, and on the 21st day, broilers and feed in each
experimental unit were weighed to determine the body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR). Broiler
mortality and temperature were recorded twice daily. From day 14 to day 21, excreta were collected twice daily. Excrement collected
from each cage was homogenized at the end of the experimental period and 200-g samples were pre-dried at 55 ◦ C for 72 h and ground
in a ball mill (Tecnal Equipamentos para Laboratório, TE-350, São Paulo, Brazil) for 5 min to obtain a fine mixture.
Then, the excrement samples were taken to the laboratory to measure the nitrogen content (method 984.13) and dry matter (DM)
(method 930.15) according to the procedures described by AOAC International (2006). Based on Joslyn (1970), insoluble acid ash and
the indigestibility ratio were calculated. The nitrogen intake (NI), nitrogen excreted (NE), nitrogen balance (NB), nitrogen utilization
efficiency (NUE), and nitrogen utilization efficiency for gain (NUEG) were analyzed. To determine the nitrogen parameters (NI, NE,
NUE, and NUEG), the average values of the five birds in each cage were used as the experimental unit, whereas for the serum pa­
rameters, the two birds closest to the mean weight in each cage were used as the experimental unit. The nitrogen intake was calculated
by multiplying the N present in diet by the DM intake. Nitrogen excreted was calculated by multiplying the total amount excreted (in
DM) by the nitrogen rate found in the excretion (also in DM). The nitrogen balance was calculated using the equation proposed by
Matterson et al. (1965).

Nbalance = Nintake – Nexcreted

At day 22, the two birds closest to the mean weight in each cage were bled via cardiac puncture for determination of plasmatic (Exp.
1) and serum (Exp. 2) uric acid (SUA) (Donsbough et al., 2010), total protein (tP), albumin (Alb) and globulin (Glob). Broilers were
fasted for two hours, and feeders were reintroduced to each pen for 30 minutes. According to Donsbough et al. (2010), this SUA
protocol can be used to determine the AA utilization in broilers fed AA-adequate and AA-deficient diets. Blood was placed into 10 ml

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R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

heparin tubes (Exp. 1) and serum tubes (Exp. 2) and samples were held on ice until centrifugation at 4000 rpm for 15 minutes. Af­
terwards, the plasma and serum were collected and separated from each bird and stored in a -40◦ C freezer. Plasma and serum in each
experiment were analyzed for SUA, tP, Albu, and Glob using commercial reagent kits (Bioclin, Belo Horizonte, Brazil). Using the SUA
and nitrogen intake, it was possible to calculate the SUANg intake and determine the correlations among these data.
BWG(g)
NEUG =
NI(g/kg)

SUA(mg/dL
SUANg =
NI(g/kg)

Where:
BWG = body weight gain;
NI = nitrogen intake;
SUA = serum uric acid.

2.4. Statistical analyses

Each pen was considered an experimental unit for all analyses. Data were analyzed by ANOVA followed by the SNK test (P<0.05)
using a randomized complete design with the GLM procedure of the SAS Institute (2009).

3. Results

3.1. Experiment 1

There was a decrease in the BWG and an increase in the FCR (P<0.05) of broilers when crude protein was decreased from 222.5 g/
kg CP to 205 g/kg CP without the addition of glutamic acid, even with an 8% increase in the eN-to-tN ratio (Table 3). No effects
(P<0.05) of the eN-to-tN ratio were observed on FI in broilers during the experiment. The treatments used in this experiment had no
effect (P<0.05) on the levels of blood parameters evaluated in this study, such as SUA, tP, Glob, and Albu in broiler chickens (Table 3).
The nitrogen intake and nitrogen balance were higher (P<0.05) in the PC and NC + Glu treatments than the values for NC without
the addition of glutamic acid (Table 4). However, the NE, NUE, NUEG, and SUANg did not differ (P<0.05) among treatments.

3.2. Experiment 2

Supplementation with glycine (NC + Gly) as a source of NEAA resulted in a decrease (P<0.05) in broiler BWG and a decrease in the
FI (Table 5), although a lower (P<0.05) FCR was observed in this treatment compared with other treatments. Broilers fed with 185 g/
kg CP without the addition of NEAA increased (P<0.05) their FI without increases in BWG, which resulted in a better FCR compared to
the control treatment. Reducing the dietary CP to 185 g/kg and individual supplementation of NEAA (Glu and Ala) or an NEAA
mixture resulted in lower FI and better FCR (P<0.05), similar to the results for the positive control (222.5 g/kg CP), compared to the a
low CP diet without the supplementation of NEAA.

Table 3
Effect of Glu supplementation and the eN-to-tN ratio on performance and blood parameters in broiler chickens from 8–21 days of age1 in the first
experiment.
Treatments eN:tN ratio PC 0.4766 NC 0.5148 NC 0.4766 + Glu SEM P-value

IBW (kg) 0.202 0.174 0.192 0.0001 0.874

FBW (kg) 1.026a 0.949b 1.004a 0.0024 0.004
BWG (kg) 0.824a 0.775b 0.812a 0.0341 0.022
FI (kg) 1.124 1.115 1.097 0.0211 0.424
FCR 1.365a 1.444b 1.351a 0.0224 0.013
SUA (mg/dL)¥ 3.010 2.960 3.930 0.1862 0.051
tP (g/dL) 2.904 3.166 3.098 0.0626 0.213
Glob (g/dL) 1.560 1.560 1.537 0.0422 0.235
Albu (g/dL) 1.508 1.606 1.561 0.0295 0.412

PC, 222.5 g/kg CP; NC, 185 g/kg CP; CP, crude protein; eN:tN, essential nitrogen:total nitrogen; IBW, initial body wight at 8 d; FBW, final body
weight at 21 d; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio; SUA, serum uric acid; tP, total protein; Glo, globulin; Albu,
albumin.
SEM, standard error of the mean.
Means on the same line, followed by different letters, differ from each other by the SNK test (P < 0.05).
Three treatments were used, with 10 replicate pens and five animals per experimental unit.
1
All birds received a common pre-starter diet from day 1–7 post-hatch, and all diets (pre-starter and starter) met or exceeded nutrient recommen­
dations for each age of chicks (Rostagno et al., (2011).

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Table 4
Effect of Glu supplementation and the eN-to-tN ratio on nitrogen utilization parameters in broiler chickens from 8 day to 21 days of age1 in the first
experiment.
Treatments eN:tN ratio PC 0.4766 NC 0.5148 NC 0.4766 + Glu SEM P-value

NE (g/bird) 19.06a 16.89b 17.65b 0.349 <0.001

NI (g/kg of diet) 46.48a 41.89b 45.00a 0.481 <0.001
NB (g/bird) 27.42a 25.00b 27.35a 0.223 <0.001
NUE 58.98 59.66 60.74 0.206 0.754
NUEG 17.88 18.51 18.05 0.155 0.229
SUANg¥ 0.0645 0.0708 0.0875 0.00410 0.057

PC, 222.5 g/kg CP; NC, 205 g/kg CP; CP, crude protein; eN:tN, essential nitrogen:total nitrogen; NE; nitrogen excreted; NI, nitrogen intake; NB;
nitrogen balance; NUE, nitrogen utilization efficiency; NUEG, nitrogen utilization efficiency to gain; SUANg, serum uric acid per gram of NI.
SEM, standard error of the mean.
¥ Serum uric acid per gram of nitrogen intake.
Means on the same line, followed by different letters, differ from each other by the SNK test (P < 0.05).
1
All birds received a common pre-starter diet from day 1–7 day post-hatch, and all diets (pre-starter and starter) met or exceeded the nutrient
recommendations for chickens of each age (Rostagno et al., 2011).
Three treatments were used, with 10 replicate pens and five animals per experimental unit.

Table 5
Effect of glycine (Gly), alanine (Ala), and glutamic acid (Glu) supplementation and the eN-to-tN ratio on performance and blood parameters in broiler
chickens from 8 to 21 days of age1 in the second experiment.
Treatments PC 0.4706 NC 0.5695 NC 0.4706 + Gly NC 0.4706 + Ala NC 0.4706 + Glu NC 0.4706 + Mix SEM P-value
eN:tN ratio

IBW (kg) 0.206 0.210 0.207 0.207 0.207 0.206 0.0001 0.361
FBW (kg) 1.065a 1.071a 0.923b 1.067a 1.044a 1.065a 0.0440 <0.001
BWG (kg) 0.859a 0.861a 0.716b 0.860a 0.837a 0.858a 0.0650 <0.001
FI (kg) 1.165bc 1.243a 0.928d 1.182b 1.128c 1.134c 0.0135 <0.001
FCR 1.357bc 1.445d 1.297a 1.375b 1.347bc 1.322ab 0.0020 <0.001
SUA (mg/dL)¥ 7.31b 7.07b 7.50b 9.81a 10.31a 11.84a 0.339 <0.001
tP (g/dL) 2.959 2.818 2.703 2.897 2.984 2.980 0.0456 0.462
Glob (g/dL) 1.281 1.290 1.143 1.241 1.330 1.337 0.0257 0.303
Albu (g/dL) 1.678 1.528 1.560 1.656 1.654 1.643 0.0244 0.414

PC, 222.5 g/kg CP; NC, 185 g/kg CP; CP, crude protein; eN:tN, essential nitrogen:total nitrogen; IBW, initial body wight at 8 d; FBW, final body
weight at 21 d; BWG, body weight gain; FI, feed intake; FCR, feed conversion ratio; SUA, serum uric acid; tP, total protein; Glob, globulin; Albu,
albumin.
SEM, standard error of the mean.
¥ Serum uric acid.
Means on the same line, followed by different letters, differ from each other by the SNK test (P < 0.05).
1
All birds received a common pre-starter diet from day 1–7 day post-hatch, and all diets (pre-starter and starter) met or exceeded nutrient recom­
mendations for chickens of each age (Rostagno et al., 2011).
2
Nonessential amino acid mixture: 50% L-Glycine, 30% Alanine, 20% Glutamate.
Six treatments were used, with 10 replicate pens and five animals per experimental unit.

In the second experiment, significant effects (P<0.05) were observed on the blood parameter SUA, with no significant effect
(P<0.05) on the other parameters analyzed (tP, Glob and Albu). A low CP diet supplemented with the Glu, Ala and NEAA mixture
resulted in a higher (P<0.05) SUA value when compared to the PC, NC and NC + Gly treatment.
Nitrogen excretion (NE) in the 185 g/kg CP group without the addition of NEAA differed significantly (P<0.05) from that in the PC
(Table 6). The NC + Gly group showed lower nitrogen excretion than the other groups. NC groups supplemented with Glu, Ala, or the
NEAA mixture showed a higher (P<0.05) NE value than the PC and NC + Gly groups. The NI was lower (P<0.05) in the NC + Gly group
than the PC grou, whereas NB was higher in this group when comparing to the groups fed low crude protein diet supplement with
NEAA. Conversely, a higher nitrogen intake was observed in groups fed the NC + Glu, Ala, or NEAA mixture, with a similar result to the
PC. The parameters NUE, NUEG, and SUANg presented significant differences (P<0.05) in this second experiment. Broilers fed a diet
with 185 g/kg CP and glycine supplementation had higher (P<0.05) NUE than broilers fed the PC and NC + Glu, Ala, or mixed NEAA
diets. Chickens fed diets with a higher eN-to-tN ratio showed a higher (P<0.05) nitrogen utilization efficiency per unit of weight than
other treatments. The SUANg of NI was higher in treatments supplemented with Glu, Ala, or the NEAA mixture, compared with the PC.

4. Discussion

Feeding broiler chickens with diets containing high levels of crude protein is not economically and environmentally viable. Diets
with a high protein content have an excess of amino acids, which will result in energy expenditure to catabolize the excess amino acids
into nitrogen (Bender, 2012; Maia et al., 2021). The nitrogen excreted will generate impacts on the environment, due to the greater

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R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

Table 6
Effect of glycine (Gly), alanine (Ala), and glutamic acid (Glu) supplementation and the eN-to-tN ratio on nitrogen utilization parameters in broiler
chickens from 8 to 21 days of age1 in the second experiment.
Treatments eN:tN ratio PC 0.4706 NC 0.5695 NC 0.4706 + Gly NC 0.4706 + Ala NC 0.4706 + Glu NC 0.4706 + Mix SEM P-value

NE (g/bird) 19.55a 18.75a 12.30b 22.74c 21.10c 21.65c 0.044 <0.001

NI (g/kg of diet) 45.54a 43.30b 38.17c 45.70a 44.13ab 45.07a 0.379 <0.001
NB (g/bird) 25.99a 24.55b 25.87a 22.96c 23.03c 23.42c 0.209 <0.001
NUE 57.10b 56.71b 68.01a 50.27c 52.36c 51.92c 0.916 <0.001
NUEG 18.87b 19.88a 18.77b 18.83b 18.98b 19.05b 0.088 <0.001
SUANg¥ 0.159c 0.163c 0.195bc 0.215ab 0.233ab 0.263a 0.0075 <0.001

PC, 222.5 g/kg CP; NC, 205 g/kg CP; CP, crude protein; eN:tN, essential nitrogen:total nitrogen; NE; nitrogen excreted; NI, nitrogen intake; NB;
nitrogen balance; NUE, nitrogen utilization efficiency; NUEG, nitrogen utilization efficiency to gain; SUANg, serum uric acid per gram of NI.
SEM, standard error of the mean.
¥ Serum uric acid per g of nitrogen intake.
Means on the same line, followed by different letters, differ from each other by the SNK test (P < 0.05).
1
All birds received a common pre-starter diet from day 1–7 day post-hatch, and all diets (pre-starter and starter) met or exceeded the nutrient
recommendations for chickens of each age (Rostagno et al., 2011).
2
Nonessential amino acid mixture: 50% L-Glycine. 30% Alanine. 20% Glutamate.
Six treatments were used, with 10 replicate pens and five animals per experimental unit.

emission of ammonia (Belloir et al., 2017), and also damages performance, as it will lead to respiratory problems for the birds (Liu
et al., 2020). The reduction of crude protein in broiler chickens’ diets seems to be a viable alternative, aiming to reduce costs attributed
to protein sources and reduce the environmental impacts resulting from nitrogen (Akinde, 2014). However, this reduction requires
essential amino acid supplementation. In some studies, a diet with a low CP content combined with EAA supplementation did not seem
to result in good production rates, so NEAA supplementation is also necessary (Wu and Li, 2022; Hou and Wu, 2017).
The effect of NEAA supplementation in low CP diets has been the focus of recent studies, especially those applying the ideal protein
concept. The synthesis of protein involves the availability of EAA and NEAA in the context of mRNA translation, which means that at
the cellular level all amino acids can be considered essential (Wu et al., 2013). In the absence of NEAA, the catabolism of EAA to
synthesize NEAA can occur. Therefore, the reduction of CP associated with essential amino acid supplementation is not enough to
improve the parameters of BWG and FCR. This corroborates the findings in the two experiments carried out, where low CP diets, even
those supplemented with EAA and without the addition of NEAA, presented a worse FCR. For this reason, it is also important to ensure
a balance between EAA and CP or EAA and NEAA (Pesti, 2009), as the absence of NEAA may prevent maximum animal performance.
Wu (2014) found that chicken diet lacking NEAA could not support maximal growth. The results of some studies have shown that
NEAA also plays an important role in regulating gene expression, cell signaling, nutrient transport and metabolism, the intestinal
microbiota, antioxidative responses, and immune responses (Wu, 2013).
Three NEAA were used in these experiments: glycine, alanine, and glutamate. Glycine is an important amino acid for birds (being
limiting in the initial phase of chicks), and its supplementation is important during the growth phase of broilers. In addition, glycine is
essential for the production of uric acid by birds (Ospina-Rojas et al., 2013; Awad, 2015). Other AAs used in the experiment were
glutamate and alanine. Glutamate is one of the most abundant AAs in the diet and protein tissues of birds, with the small intestine,
liver, pancreas, skeletal muscle and brain as the sites of catabolism (He et al., 2021). The role of alanine is not clear in poultry nutrition,
but some studies suggest that the use of alanine can improve parameters such as average daily gain and feed conversion in broiler
chickens (Qi et al., 2018; Jacob et al., 1991).
The first experiment found that dietary CP reduction supplemented with essential crystalline amino acids to meet AA requirements
may not be enough to improve the weight gain of broiler chickens when compared to higher CP diets (Ospina-Rojas et al., 2013). Pesti,
(2009) and Dean et al. (2006) mentioned that the growth rate can decrease and the FCR increase in a low CP diet, even when sup­
plemented with EAA, specifically due to the lack of non-specific nitrogen to synthesize NEAA. In this study, all diets were supple­
mented with EAA, even when the crude protein was reduced, and this reduction without the addition of glutamic acid decreased the
BWG and worsened the FCR in the first experiment when comparing the NC treatment to the other treatments utilized. The supple­
mentation of 28 g/kg Glu was sufficient to recover 3% of the BWG and improved the FCR by 9% in low CP diets.
Previous studies have reported that the application of glutamine enhanced growth performance, regulated immune responses, and
altered tight junction protein expression (Luquetti et al., 2016; Oxford and Selvaraj, 2019). Another function of Glu is that it plays an
essential role in maintaining the intestinal health of broiler chickens and reduces intestinal permeability in animals (Teng et al., 2021).
This helps explain the results when we add glutamine to the diet, because with good intestinal health, animals can perform well when
presenting an intestinal environment favorable to good digestion and the absorption of nutrients, thus improving BWG, FI, and FCR
results (Sakamoto et al., 2006; Wu et al., 2019).
Some experiments with broiler chickens have been carried out to study the effect of adding NEAA to low CP diets, and the results
showed a positive response to high non-essential nitrogen levels by decreasing the FI, moderately reducing the growth rate, and
improving the FCR (Heger, 2003; Parr and Summers, 1991; Han et al., 1992). He et al. (2021) recommended that Glu must therefore be
taken into consideration when the optimum AA composition for feeding poultry is formulated. One of the reasons for this recom­
mendation is that, among the crystalline amino acids, glutamate seems to be the most effective source of non-specific nitrogen (Allen
and Baker, 1974). According to Walker and van der Donk (2016), glutamate donates nitrogen to the amino group of all amino acids

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R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

through the action of glutamate transaminases. There is evidence that Glu must be provided in the diets of broilers for their maximum
growth, as well as optimum development and health (Hou et al., 2015; Hou and Wu, 2017; Kidd, Kerr, 2000; Maruyama et al., 1975).
Nitrogen efficiency is a parameter that should be analyzed to verify the possible effects that a diet with greater or lesser CP content
could have on the environment due to greater or lesser nitrogen excretion (Hilliar et al., 2020). In this experiment, reducing CP from
222.5 to 205 g/kg CP in the broiler diet also reduced nitrogen intake and nitrogen balance. However, when glutamic acid was added to
the broiler diet with 205 g/kg CP, NI and NB improved by 3% and 2%, respectively, these results being similar to those obtained in the
PC. Bregendahl et al. (2002) observed that birds fed a low CP diet had lower nitrogen retention than those fed high-CP diets. In
contrast, Chrystal et al. (2019) reported that reductions in dietary protein from 210 g/kg to 165 g/kg day did not affect N retention, but
there was an increase in energy utilization as the ME:GE ratio increased.
Aletor et al. (2000) found a higher NUE when the CP level was reduced from 225 to 153 g/kg CP. Glu is often used to balance the
dietary nitrogen content in studies involving poultry (Baker and Han, 1994). No effect was observed for SUA, Alb and Glob; however,
the SUA/NI ratio tended to be higher in birds fed NC + Glu, probably due to the higher amount of crystalline amino acids in the diet.
In the second experiment, we observed that the addition of Gly to a diet with a reduction in CP to 180 g/kg resulted in a 14%
decrease in BWG and a 23% decrease in FI. In this study, a total of 49.8 g/kg of glycine + serine was provided, resulting in 3.3 g/day of
glycine. Dean et al. (2006) reported that the proportion of Gly + Ser should be 24.4 g/kg to support the optimal growth of broilers in
diets with 6% lower CP without adding other amino acids. Waguespack et al. (2009) suggested a minimum of 21 g/kg of Gly + Ser for
maximum performance in broilers fed a low-CP, corn-soybean meal diet supplemented with Lys, Met, and Thr. Glycine is essential for
feather development, representing 15% of feather protein and 30% of the AAs present in collagen, and it is an important component of
the uric acid molecule. However, in the present study there is no doubt that the high inclusion of glycine can lead to a toxic effect on
broilers. Adding Gly to low CP diets with a high proportion of crystalline amino acids improved performance but not completely to
overcome the adverse effects of a high quantity of crystalline amino acids in low CP diets (Namroud et al., 2010). Harn et al. (2019)
evaluated the effect of reducing dietary protein by up to 3% of commercial levels with the addition of essential amino acids (including
glycine) and found no reductions in performance parameters in either the starter or finisher phase.
The use of Glu, Ala, or the NEAA mixture gave better results when compared to the NC not supplemented with NEAA. Birds fed with
185 g/kg CP without the addition of NEAA had a higher FI and a worse FCR than those fed with PC and NC supplemented with Glu, Ala
or the NEAA mixture. The levels of NEAA in the diet can influence FI (Aftab et al., 2006), whereby the reduction of CP can limit the
presence of NEAA in the diet and thus influence the FI. Chrystal et al. (2020) reported a worse FCR in broiler chickens fed a diet with
protein reduced to 165 g/kg CP. According to Hilliar et al. (2020) reducing CP below 190 g/kg in the growing period reduces BWG and
FCR. This effect can be explained by the physiology of growing animals, where chicks are more reactive to dietary changes in CP than
finishing animals (Kriseldi et al., 2017), and the presence of NEAA in a low CP diet can be limiting.
In our experiment, NEAA supplementation with Glu, Ala or a mixture was efficient to recover performance. Awad et al. (2015)
reported that adding a mixture of NEAA (Gly, Glu, Pro, Ala and Asp) to a low crude protein diet improved broiler performance. This
effect can be explained by the mechanisms of action of glutamine, which is extremely important for cellular metabolic processes, and
essential for the growth of most cells and tissues (Pierzynowski et al., 2001). In situations of high protein degradation, glutamine acts
as a metabolic regulator to increase synthesis and reduce protein catabolism. Periods of rapid tissue growth are related to this increased
protein degradation, where endogenous synthesis may not be sufficient (Lobley et al., 2001). In addition, glutamine is considered the
major energy substrate for rapidly proliferating cells, such as enterocytes and active lymphocytes (Dewitt et al., 1999).
According to Zavarize et al. (2011), glutamine supplementation in the starter phase of broilers has positive effects on intestinal
maturation, a period accompanied by morphological and physiological changes, allowing for an increase in digestion and absorption
surface area, and consequently improving performance parameters during the starter period of digestive tract development. In this
experiment, individual Ala supplementation in diets with 185 g/kg CP resulted in better BWG and FCR, similar to the PC (222.5 g/kg
CP). However, Award et al. (2015) observed that individual Ala supplementation in a low CP diet (162 g/kg CP) had no positive effect
on BWG or FCR.
The best FCR with an eN-to-tN ratio of 0.4706 was a result of lower FI, once we did not have effect on weight gain. The effect on FI
was probably a result of the lower need for EAA utilization to synthesize NEAA, once non-specific nitrogen was supplied in the diet
through supplementation with Glu, Ala or the NEAA mixture. In contrast to our results, Hussein et al. (2001) added 2.8 or 5.5% of L-
glutamate to low CP diets and observed that birds fed with those diets were not able to recover performance, suggesting that even a
protein reduction from 22.6% to 17.6% non-specific nitrogen was not a limiting factor.
Decreasing dietary crude protein tends to have a positive effect on N utilization efficiency by increase NUE. The results of this
experiment show that a low CP diet supplemented with Glu, Ala, or an NEAA mixture had a lower NB and NUE than the NC diets
without the supplementation of NEAA. However, the addition of Gly to a low CP diet improved the NB, showing a higher NUE than in
the PC and NC groups. According to Siegert and Rodehutscord, (2019), the supplementation of Gly in the diet increased the nitrogen
utilization efficiency in diets with adequate EAA. However, Hofmann et al. (2019) mentioned that the supplementation of Glu in­
creases NUE more than other NEAA combinations, such as Asp+Asn and Glu+Gln. Wu (2013) attributed these differences to the
different contributions of nitrogen in the diet; free amino acids are metabolized more rapidly than nitrogen in intact protein. Si et al.
(2004) reported that decreasing dietary CP from 200 to 180 g/kg decreased nitrogen content in the excreta by 13% with no negative
effect on BWG or the FCR of broilers from 1 to 21 d of age. This corroborates the results found in the current study, as diets with
185 g/kg CP + Gly led to lower excreted nitrogen.
The evaluation of plasma biochemistry in poultry is essential for the identification of metabolic changes occurring due to several
factors, including the type of diet. A hyperproteic diet can elevate serum uric acid values as a result of increased protein catabolism
(Rajman et al., 2006). Uric acid is the main nitrogenous waste product of poultry, produced and secreted mainly by the liver, and to a

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R.C. Maia et al. Animal Feed Science and Technology 315 (2024) 116010

lesser extent by the kidneys and pancreas. Uric acid concentrations can vary with age (with higher values in younger animals), with the
diet (being higher in foods with higher protein content), or with physiological or metabolic changes (Capitelli and Crosta, 2013).
It became clearer when comparing the SUA and SUA/NI for treatments supplemented with NEAA. These produced more SUA per g
of nitrogen intake. The NC had a better NUEG than other treatments because there was no effect on BWG when the eN:tN ratio
increased. Namroud et al. (2008) showed that a reduction in dietary CP decreased uric acid excretion; however, Waguespack et al.
(2009) reported that the addition of Gly decreased SUA and SUN concentrations in broilers fed 1.35% Lys but increased the SUA
concentration in broilers fed 1.26% Lys.
Birds fed the low protein diet (NC) or NC + individual NEAA diets had lower serum uric acid levels when compared to the treatment
without protein reduction (standard commercial diet) (Awad et al., 2015). According to the same authors cited above, this behavior
can be attributed to the lower intake of AAs in birds fed a low CP diet, since uric acid is the end product of protein catabolism.

5. Conclusion

Broiler chickens fed a low crude protein diet with an essential nitrogen:total nitrogen ratio of 0.4706 supplemented with EAA and
NEAA showed better performance parameters (such as BWG and FCR) than those fed with a low crude protein diet without NEAA.
Nitrogen utilization efficiency seems to be increased by reducing crude protein down to 37.5 g/kg, but adding non-essential amino
acids.

CRediT authorship contribution statement

Bruno Damaceno Faria: Investigation. Luiz Fernando Teixeira Albino: Funding acquisition, Investigation, Supervision. Arele
Arlindo Calderano: Writing – review & editing. Rosana Cardoso Maia: Conceptualization, Data curation, Formal analysis, Inves­
tigation, Methodology, Writing – original draft. Rafael Sousa Ferreira: Writing – review & editing. Horácio Santiago Rostagno:
Funding acquisition, Methodology, Project administration, Supervision. Diego Ladeira da Silva: Investigation. Thaisa Pereira de
França: Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgments

The authors wish to thank the Universidade Federal de Viçosa (Viçosa, Brazil) and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq; Brasília, Brazil) for financial support.

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