Animal Feed Science and Technology 273 (2021) 114753

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Animal Feed Science and Technology

journal homepage: www.elsevier.com/locate/anifeedsci

Combination of non-protein nitrogen and N‑carbamylglutamate

supplementation improves growth performance, nutrient
digestibility and carcass characteristics in finishing pigs fed low
protein diets
P.L. Li a, b, F. Wu c, Y.M. Wang a, b, C.P. Wang c, J.Y. Zhou a, b, X.F. Zeng a, b, S.
Y. Qiao a, b, *
a
State Key Laboratory of Animal-Nutrition, College of Animal Science and Technology, Ministry of Agriculture Feed Industry Centre, China
Agricultural University, Beijing, 100193, China
b
Beijing Key Laboratory of Biological Feed Additive, China Agricultural University, Beijing, 100193, China
c
Beijing Animore Sci. & Tech. Co. Ltd., Beijing, 100193, China

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

Keywords: This study was conducted to determine whether supplemental non-protein nitrogen (NPN) and/or
Finishing pigs N-carbamylglutamate (NCG) in the low protein diet enhance the growth performance, nutrient
Low protein diet digestibility and carcass characteristics of finishing pigs. A total of 150 (Duroc boar× (Landrace
N-carbamylglutamate
boar × Yorkshire) sow) finishing pigs (initially body weight 74.3 ± 3.8 kg) were blocked by
Non-protein nitrogen
Performance
weight and sex, and allotted to one 5 treatments with 6 replicate pens (3 barrows and 2 gilts per
pen). Diets were normal protein diet (NP, crude protein (CP) 136.4 g/kg), low protein diet (LP, CP
111.4 g/kg), LP supplemented with 0.8 g/kg NCG (LNCG, CP 111.8 g/kg), LP supplemented with
7.1 g/kg NPN (LNPN, CP 119.8 g/kg), and LP supplemented with 7.1 g/kg NPN and 0.8 g/kg
NCG (LNN, CP 120.3 g/kg) and fed to pigs for 40 days. At the end of the experiment, 30 pigs (one
barrow per pen) were slaughtered to evaluate carcass traits and meat quality. Compared to the LP
group, the combination of NPN and NCG supplementation significantly increased (P < 0.05) the
average daily gain (ADG) of finishing pigs. The coefficient of apparent total tract digestibility
(CATTD) of gross energy (GE), crude protein (CP) and acid detergent fiber (ADF) in LP group were
lower (P < 0.05) than that in the NP group, while LNN group significantly increased (P < 0.05)
the CATTD of GE, CP, ether extraction (EE), neutral detergent fiber (aNDF) and ADF when
compared with LP group. Similarly, the concentrations of arginine, isoleucine, threonine, glycine,
serine, tyrosine, and citrulline in the serum of pigs fed LNN diet were higher (P < 0.05) than that
in those pigs fed LP diet. The carcass traits and meat quality did not differ between the NP group
and the LP group, but the combination of NPN and NCG supplementation in LP diet significantly
increased (P < 0.05) the concentrations of intramuscular fat. Compared to NP group, LP group

Abbreviations: AA, amino acids; ADF, acid detergent fiber; ADFI, average daily feed intake; ADG, average daily gain; CATTD, coefficient of
apparent total tract digestibility; BW, body weight; CP, crude protein; CPS, carbamoyl phosphate synthase; DM, dry matter; F/G, feed to gain ratio;
GE, gross energy; IMF, intramuscular fat; NCG, N-carbamylglutamate; aNDF, neutral detergent fiber assayed with a heat stable amylase and
expressed inclusive of residual ash; NE, net energy; NEAA, non-essential amino acids; NPN, non-protein nitrogen; PUFA, polyunsaturated fatty acids.
* Corresponding author at: State Key Laboratory of Animal-Nutrition, College of Animal Science and Technology, Ministry of Agriculture Feed
Industry Centre, China Agricultural University, Beijing, 100193, China.
E-mail address: qiaoshiyan@cau.edu.cn (S.Y. Qiao).

https://doi.org/10.1016/j.anifeedsci.2020.114753
Received 26 November 2019; Received in revised form 1 November 2020; Accepted 9 November 2020
Available online 29 December 2020
0377-8401/© 2020 Published by Elsevier B.V.
P.L. Li et al. Animal Feed Science and Technology 273 (2021) 114753

increased (P < 0.05) the concentration of C16:0 and decreased (P < 0.05) the concentrations of
C20:4, C22:6 and polyunsaturated fatty acid (PUFA) in the M. longissimus lumborum of finishing
pigs, and LNN group further decreased (P < 0.05) the concentrations of C18:2 and PUFA when
compared with the LP group. This study indicates that dietary supplementation with both NPN
and NCG has important complementary advantages in improving the growth performance and
meat quality of finishing pigs fed low protein diet balanced with amino acids.

1. Introduction

Reducing dietary crude protein (CP) with free amino acids (AA) supplementation is gaining increasing attention, because compared
to traditional diets, the low protein diet allows reduction of nitrogen (N) excretion and promotion of intestinal health while main­
taining performance of pigs (Wang et al., 2018a). Through supplementing some limiting amino acids (mostly lysine, threonine,
methionine and tryptophan), dietary CP content in cereal-soybean meal-based diet can be reduced by approximately 2%–4% from the
recommendations of NRC (1998) without reducing N retention and performance of pigs (Figueroa et al., 2003; Kerr et al., 2003).

Table 1
Ingredient composition of experimental diets (g/kg, as-fed basis).
Item NP LP LNCG LNPN LNN

Ingredients
Corn 774.8 811 800.2 804.6 803.4
Soybean meal 140 44 44 44 44
Wheat bran 57 110 110 110 110
Soybean oil – – – 2.0 2.4
Limestone 6.6 7.4 7.4 13.2 13.2
Dicalcium phosphate 8.5 8.5 8.5 – –
Salt 4.0 4.0 4.0 4.0 4.0
Vitamin and mineral premixa 5.0 5.0 5.0 5.0 5.0
L-lysine-HCl 2.7 5.3 5.3 5.3 5.3
DL-methionine 0.3 1.0 1.0 1.0 1.0
L-threonine 0.9 1.9 1.9 1.9 1.9
L-tryptophan 0.1 0.5 0.5 0.5 0.5
L-valine 0.1 1.4 1.4 1.4 1.4
Diammonium phosphate – – – 7.1 7.1
N-carbamylglutamate – – 0.8 – 0.8
Analyzed composition
Dry matter 878.5 880.0 880.6 878.5 881.3
Crude proteinb 136.4 111.4 111.8 119.8 120.3
Ether extract 28.8 29.0 28.8 30.4 31.0
aNeutral detergent fiber 162.5 182.0 181.5 184.3 183.4
Acid detergent fiber 46.3 47.6 47.0 47.4 47.6
Ash 38.6 37.6 36.8 37.8 36.1
Lysine 8.7 9.2 9.3 9.3 9.3
Methionine 2.8 3.0 2.9 3.0 2.9
Threonine 5.6 5.5 5.5 5.4 5.5
Tryptophan 1.5 1.5 1.5 1.4 1.5
Valine 5.8 5.6 5.7 5.6 5.6
Arginine 7.2 5.2 5.3 5.2 5.3
Calculated composition
Net energy, MJ/kg 10.37 10.37 10.36 10.36 10.36
Standardized ileal digestible AAc
Lysine 7.3 7.3 7.3 7.3 7.3
Methionine + cysteine 4.2 4.2 4.2 4.2 4.2
Threonine 4.6 4.6 4.6 4.6 4.6
Tryptophan 1.3 1.3 1.3 1.3 1.3
Valine 4.8 4.8 4.8 4.8 4.8

AA, amino acids; NCG, N-carbamylglutamate; NP, normal diet (136.4 g/kg CP); NPN, non-protein nitrogen, diammonium phosphate was used as the
source of non-protein nitrogen; LNCG, low protein diet supplemented with NCG; LNN, low protein diet supplemented with NPN and NCG; LNPN, low
protein diet supplemented with NPN; LP, low protein diet (111.4 g/kg CP).
a
Premix provided per kilogram of diets: vitamin A, 3500 IU as vitamin A acetate; vitamin D, 1000 IU as vitamin D3; vitamin E, 10 IU as DL-
α-tocopheryl acetate; vitamin K3, 2.1 mg; vitamin B1, 0.7 mg; vitamin B2, 2.1 mg; vitamin B6, 1.0 mg as pyridoxine hydrochloride; vitamin B12,
7.0 μg; nicotinic acid, 14.0 mg; pantothenic acid, 10.0 mg as calcium pantothenate; biotin, 30.0 mg; folacin, 0.5 mg; Mn, 13 mg as manganese sulfate;
Fe, 75 mg as ferrous sulfate monohydrate; Zn, 75 mg as zinc sulfate; Cu (CuSO4⋅5H2O), 30 mg; I, 0.48 mg as calcium iodate; and Se, 0.45 mg as
sodium selenite.
b
The calculation of crude protein (N × 6.25) included the nitrogen supplied by non protein nitrogen.
c
Values for standardized ileal digestible AA were calculated from the analyzed amino acid content and standardized ileal amino acid digestibility of
the ingredients according to NRC (2012).

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P.L. Li et al. Animal Feed Science and Technology 273 (2021) 114753

However, there were studies showed that feed efficiency decreased when dietary CP protein level was further reduced, which indicated
that the insufficient supply of nitrogen or non-essential amino acids (NEAA) may be a limiting factor (Powell et al., 2011; Gloaguen
et al., 2014).
Recent studies have shown that non-protein nitrogen (NPN) in the form of ammonium salts was as efficient as N from amino acid-N
sources (i.e., intact protein or AA) in suppling NEAA for improving growth performance and nitrogen retention of pigs fed semi-
purified diets severely deficient in NEAA (Mansilla et al., 2017, 2018). Thus, it is possible that inclusion of ammonia-nitrogen may
also enhance the growth performance of pigs fed low protein diets composed by common feed ingredients.
In mammals, ammonia forms carbamylphosphate with bicarbonate in the initial step of the urea cycle catalyzed by carbamoyl
phosphate synthetase I (CPS-I), an enzyme that can be activated by N-carbamylglutamate (NCG) (Meijer et al., 1985; Wu, 1997; Wu
et al., 2004). Moreover, Frank et al. (2007) reported that oral NCG supplementation increased protein synthesis in skeletal muscle of
piglets, and our previous studies also showed that NCG supplementation improved the growth performance, nutrient digestibility,
carcass traits and intestinal function of pigs (Zeng et al., 2015; Ye et al., 2017; Wang et al., 2019a). Thus, we hypothesize that sup­
plementing NCG to low protein diets will increase the utilization of ammonia nitrogen to support a better productive performance.
Therefore, the objective of this study was to determine the effects of dietary supplementation with NPN or in combination with NCG
on the growth performance, nutrient digestibility, and carcass characteristics of finishing pigs fed low protein diets.

2. Materials and methods

All experimental procedures and animal care were approved by the China Agricultural University Animal Care and Use Committee
(Beijing, China; ID: SKLAB-B-2010-003). Experiment was conducted at the Pig Research Facility at the Swine Nutrition Research
Centre of the National Feed Engineering Technology Research Centre (Chengde, Hebei Province, China).

2.1. Animals, diets and experiment design

A total of 150 pigs (Duroc boar × (Landrace boar × Yorkshire) sow; 90 barrows and 60 gilts), with an average initial body weight
(BW) of 74.3 ± 3.8 kg at approximately130 days of age, were blocked by weight and sex and assigned randomly to 5 dietary treatments
with 6 replicates (3 barrows and 2 gilts per replicate pen). The dietary treatments were normal protein diet (NP, CP 136.4 g/kg), low
protein diet (LP, CP 111.4 g/kg), LP plus 0.8 g/kg NCG (LNCG, CP 111.8 g/kg), LP plus 7.1 g/kg NPN (LNPN, CP 119.8 g/kg) or LP
plus 7.1 g/kg NPN and 0.8 g/kg NCG (LNN, CP 120.3 g/kg) and fed to pigs for 40 days. The NCG (purity ≥ 970 g/kg) was provided by
Beijing Animore Sci. & Tech. Co., Ltd. (Beijing, China) and NPN was provided in the form of diammonium phosphate (purity ≥ 990 g/
kg) purchased from Beijing Kangpu Huiwei Co., Ltd. (Beijing, China). On the premise of the same content of calcium and phosphorus in
different diets, the addition of NPN was maximized. The crystalline amino acid of lysine, methionine, threonine, tryptophan, and
valine were added to all the diets to balance and meet the requirements of NRC (2012). The ingredients and nutrient compositions of
experimental diets are shown in Table 1. In the last week of experiment, diets were supplemented with 3 g/kg Cr2O3 as an indigestible
marker to determine the nutrient digestibility of diets.
During the experimental period, pigs were placed in partially steel-slatted concrete floored pens and each pen was equipped with a
stainless-steel feeder and a nipple drinker. Feed and water were available at all times. The body weight of individual pig was recorded
at the beginning and end of the experiment. The average daily gain (ADG), average daily feed intake (ADFI), and feed to gain ratio (F/
G) were calculated on a pen basis.

2.2. Sample collection

Fresh fecal samples were collected in each pen from 0800 to 1600 h for the last 3 days of the experiment by hand grab-sampling
from carefully cleaned pen floors to determine the coefficient of apparent total tract digestibility (CATTD) of dietary nutrients. Fecal
samples were pooled together by pen and stored at − 20 ◦ C until further analyses. At the end of the experiment, one barrow from each
pen, whose BW was close to the average BW of their pen, was selected and blood sampled after an overnight fast. Pigs were bled via
jugular vein puncture using a 0.9-mm-diameter needle into uncoated vacutainer tubes (Becton Dickinson Vacutainer Systems, Franklin
Lakes, NJ, USA). Blood samples were quickly centrifuged at 3000×g (Heraeus Biofuge 22R Centrifuge, Hanau, Germany) for 10 min
and the serum was stored at − 20 ◦ C until analyzed for serum AA and urea nitrogen. After blood collection, the selected barrows were
transported to Tangshan shineway foods Co. Ltd (Tangshan, Hebei province, China) for slaughter to measure carcass characteristics
and meat quality.

2.3. Carcass characteristics and meat quality measurements

Carcass weight was immediately recorded following slaughter and the dressing percentage was determined as the ratio of carcass
weight to live weight. The carcass was split and then the left side was cut between the 10th and 11th rib to allow measurement of loin-
eye area, tenth rib fat depth, 45-min and 24-h pH (hand-held pH meter, Model 2000, VWR Scientific Products Co., South Plainfield, NJ,
USA). Carcass fat-free lean percentage was calculated using the equation of the National Pork Producers Council (Burson and Berg,
2001): Fat-free lean, kg/100 kg = ([8.588 + (0.465 × hot carcass weight, lb) – (21.896 × 10th rib backfat, inch) + (3.005 × 10th loin
eye area, inch2)] ÷ hot carcass weight, lb) × 100. The loin (M. longissimus lumborum) samples were also taken for analyzing meat
quality, and the composition of fatty acid and AA. Drip loss was calculated by hanging a loin muscle sample (5 cm × 8 cm × 2.5 cm,

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P.L. Li et al. Animal Feed Science and Technology 273 (2021) 114753

about 100 g) in an inflated and closed plastic bag for 24 h at 4 ◦ C (King et al., 2000). The CIELab L* (lightness), a* (redness), and b*
(yellowness) color was determined by Colorimeter (Chromameter, CR410, Minolta, Japan) after blooming for 15 min.

2.4. Biochemical analyses

Experimental diets and feces were analyzed for dry matter (DM, Method 930.15, AOAC, 2006), and ether extract (EE, Method
920.39, AOAC, 2006). Determination of CP content in diets and feces were based on total nitrogen content by Kjeldahl (Method
984.13, AOAC, 2006) and calculated by multiplying the total nitrogen by 6.25. It should be noted that the N content used to calculate
the CP content includes both the amino acid-N and the nitrogen supplied by NPN. aNeutral detergent fiber (aNDF) and acid detergent
fiber (ADF) were determined using filter bags and fiber analyzer equipment (Fibre Analyzer, Ankom Technology, Macedon, NY, USA)
following a modification of the procedures of van Soest et al. (1991). The concentration of aNDF was analyzed using heat stable
α-amylase and expressed inclusive residual crude ash. ADF expressed inclusion of crude ash. The gross energy (GE) in diets and feces
were analyzed using an Isoperibol Calorimeter (Parr 6300 Calorimeter, Moline, IL, USA).
The AA content in the ingredients and diets (except methionine, cystine, and tryptophan) were detected by ion-exchange chro­
matography using a Hitachi L-8800 AA Analyzer (Tokyo, Japan) following acid hydrolysis with 6 N HCl. Dietary methionine and
cysteine were measured by acid hydrolysis with HCl after an oxidation step for quantification of total sulphur, and tryptophan was
determined using reverse-phase high performance liquid chromatography (HPLC) (Waters 2690, Milford, MA, USA) after an alkaline
hydrolysis at 120 ◦ C for 16 h. The concentrations of chromium in diets and feces were determined using a polarized Zeeman Atomic
Absorption Spectrometer (Hitachi Z2000, Tokyo, Japan) after nitric acid-perchloric acid wet ash sample preparation according to the
procedure of Williams et al. (1962).
Serum amino acid concentrations were determined by reverse-phase HPLC after derivatization with o-phthaldialdehyde, as
described by Dai et al. (2014). Serum urea nitrogen concentration was determined with a blood urea nitrogen color test kit (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China). The M. longissimus lumborum samples were freeze-dried for 96 h, weighed, and
ground in liquid nitrogen. The CP and intramuscular fat (IMF) and AA were analyzed by the same methods as diet and feces. Fatty acid
composition was determined by gas chromatography (Agilent 6890 N, Santa Clara, CA, USA) described by Ye et al. (2017). The
concentration of individual fatty acid was expressed as a percentage of total fatty acids.

2.5. Statistical analysis

Data were statistically analyzed by ANOVA using the General Linear Model (GLM) procedure of SAS (2008). For the growth
performance and CATTD of nutrients, pen served as the experimental unit, and the individual pig (barrow) was the experimental unit
for other parameters. Treatment means were compared using Tukey’s multiple comparison test. Differences were considered to be
significant at P < 0.05. Results are expressed as the means and standard error of the mean.

3. Results

3.1. Growth performance

There were no differences in the final BW, ADFI and F/G among treatments (Table 2). The ADG of pigs in LP group were 5.1 % lower
than that in NP group, but the difference was not statistically significant. Compared to the LP group, adding NPN or NCG alone to the
LP diet did not affect the ADG, but the combination of NPN and NCG supplementation in the LP diet significantly increased (P < 0.05)
the ADG of finishing pigs by 7.8 %. LNPN diet had a significantly lower ADG than the NP diet (P < 0.05).

Table 2
Effects of dietary supplementation with NPN and/or NCG on the growth performance in finishing pigs throughout the 40 days experiment.
Item NP LP LNCG LNPN LNN SEM Effect (P-values)

Number of pensd 6 6 6 6 6
Initial BW, kg 74.4 74.4 74.2 74.3 74.1 3.81 0.998
Final BW, kg 107.9 106.1 107.6 105.1 108.3 4.42 0.940
ADG, g 836ab 793bc 833ab 767c 855a 21.1 <0.001
ADFI, g 2704 2554 2645 2350 2691 141.2 0.112
F/G 3.23 3.22 3.18 3.06 3.14 0.114 0.551

ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; F/G, feed to gain ratio; NCG, N-carbamylglutamate; NP, normal diet
(136.4 g/kg CP); NPN, non-protein nitrogen, diammonium phosphate was used as the source of non-protein nitrogen; LNCG, low protein diet sup­
plemented with NCG; LNN, low protein diet supplemented with NPN and NCG; LNPN, low protein diet supplemented with NPN; LP, low protein diet
(111.4 g/kg CP); SEM, the standard error of the mean.
a− c
Means within a row without a common superscript letter differ (P < 0.05).
d
There were 6 pen replicates per treatment (with 3 barrows and 2 gilts each pen) in the present study.

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3.2. Nutrient digestibility

Although supplementation NPN or NCG alone in low protein diet did not affect the CATTD of nutrients (Table 3), but LNN group
significantly increased (P < 0.05) the CATTD of GE, CP, EE, aNDF and ADF when compared with LP group. LNPN group had a
significantly lower (P < 0.05) CATTD of DM than LNN diets and a significantly lower (P < 0.05) CATTD of GE, CP and ADF than NP
and LNN groups.

3.3. Serum amino acids and urea nitrogen

The concentrations of arginine, isoleucine, threonine, glycine, serine (Table 4), tyrosine, and citrulline in the serum of pigs fed LNN
diet were higher (P < 0.05) than that in those pigs fed LP diet. Supplementing NPN and/or NCG in the LP diet did not affect serum urea
nitrogen, but LNPN group had higher (P < 0.05) urea nitrogen in serum compared with LNCG and LNN group.

3.4. Carcass traits and meat quality

The carcass traits and meat quality did not differ between the NP group and the LP group (Table 5). Compared with the LP group,
LNN group significantly increased (P < 0.05) the concentrations of intramuscular fat. The tenth rib fat depth was higher and the fat-
free lean percentage was lower in LNPN group (P < 0.05) than that in LNCG group.

3.5. Amino acid and fatty acid compositions in the M. Longissimus lumborum

As shown in Table 6, the concentrations of aspartate in LNN group and glutamate in LP and LNPN group were lower (P < 0.05) than
that in the NP group in the M. longissimus lumborum of finishing pigs.
Supplying NPN or NCG alone in the LP diet did not affect the fatty acid compositions (Table 7), but the combination of NPN and
NCG supplementation further decreased (P < 0.05) the concentrations of C18:2 and PUFA when compared with the LP group.

4. Discussion

In the present study, compared to the NP group, the growth performance of finishing pigs fed LP diets was not significantly
decreased, but adding NPN to the LP diet decreased the ADG of finishing pigs, which is inconsistent with our expectations and also the
recent studies by Mansilla et al. (2017, 2018) who found that NPN can be used as N source to improve growth performance when diets
were severely deficient in NEAA. The reason for this discrepancy may be the difference in the degree of dietary NEAA deficiency
between the present experiment and the study of Mansilla et al. (2015, 2017) (slight deficiency vs. serious deficiency). Because
previous studies have found that when the dietary nitrogen was just supplied in the form of ten essential amino acids at their minimum
levels of requirement, the addition of ammonium salts or even urea to such a ration could induces a remarkable enhancement in the
rate of gain in weanling rats (Rose et al., 1949). Moreover, direct supplementation of NEAA could also improve the growth perfor­
mance and N utilization in pigs fed low protein diets (Powell et al., 2011; Gloaguen et al., 2014). In this study, the concentration of
NEAA in the serum of finishing pigs was not improved by adding NPN to the LP diet, which indicates that NPN may not be an effective
N source for NEAA synthesis in low protein diets that were not severely deficient in NEAA. Therefore, based on previous studies and the
results of present experiment, we speculate that when dietary NEAA is not seriously deficient, the ability of NPN to synthesize NEAA is
limited, and it can hardly improve or may even decrease the growth performance of pigs fed low protein diets.
Interestingly, the results of this study showed that the LNN group significantly increased the growth rate of finishing pigs when
compared with both LP and LNPN group, which confirmed our hypothesis that NCG could promote the utilization of NPN and then
improve the growth performance of pigs. As an analogue of N-acetylglutamate, NCG can also activate CPS-I and help ammonia

Table 3
Effects of dietary supplementation with NPN and/or NCG on the coefficient of apparent total tract digestibility of nutrients in finishing pigs.
Item NP LP LNCG LNPN LNN SEM Effect (P-values)

Number of pensc 6 6 6 6 6
Dry matter 0.833ab 0.822ab 0.824ab 0.818b 0.833a 0.0049 0.021
Gross energy 0.821a 0.806b 0.812ab 0.801b 0.822a 0.0047 <0.001
Crude protein 0.813a 0.770b 0.780b 0.762b 0.809a 0.0095 <0.001
Ether extract 0.429b 0.418b 0.435b 0.466ab 0.501a 0.0204 <0.001
aNDF 0.552ab 0.535b 0.552ab 0.543ab 0.579a 0.0130 0.029
ADF 0.450a 0.383b 0.407b 0.395b 0.447a 0.0123 <0.001

ADF, acid detergent fiber; aNDF, neutral detergent fiber assayed with a heat stable amylase and expressed inclusive of residual ash; NCG, N-car­
bamylglutamate; NP, normal diet (136.4 g/kg CP); NPN, non-protein nitrogen, diammonium phosphate was used as the source of non-protein ni­
trogen; LNCG, low protein diet supplemented with NCG; LNN, low protein diet supplemented with NPN and NCG; LNPN, low protein diet
supplemented with NPN; LP, low protein diet (111.4 g/kg CP); SEM, the standard error of the mean.
a− b
Means within a row without a common superscript letter differ (P < 0.05).
c
There were 6 pen replicates per treatment (with 3 barrows and 2 gilts each pen) in the present study.

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Table 4
Effects of dietary supplementation with NPN and/or NCG on the serum amino acids (nmol/mL) and urea nitrogen (mmol/L) of finishing pigs.
Item NP LP LNCG LNPN LNN SEM Effect (P-values)

Number of animalsd 6 6 6 6 6
Arginine 283a 199c 230bc 192c 253ab 17.1 <0.001
Histidine 92a 63ab 71ab 58b 82ab 10.1 0.001
Isoleucine 117a 43c 56bc 48c 83b 11.3 <0.001
Leucine 248a 214ab 209ab 189b 224ab 15.9 0.021
Lysine 312 415 429 337 455 56.4 0.069
Methionine 49ab 68a 68a 45b 69a 7.1 <0.001
Phenylalanine 97a 69b 70b 66b 84ab 7.0 <0.001
Threonine 204b 182b 235ab 146b 320a 30.2 <0.001
Tryptophan 59 53 64 46 66 10.1 0.261
Valine 271 258 276 247 330 33.9 0.172
Alanine 475 634 557 544 521 75.8 0.328
Aspartate 25 20 18 17 22 3.1 0.112
Asparagine 67a 52ab 51ab 43b 62a 6.0 0.011
Glutamate 220b 266ab 281ab 210b 342a 33.2 <0.001
Glutamine 507 558 534 516 523 40.1 0.722
Glycine 951b 927b 963b 1153ab 1311a 89.9 <0.001
Serine 150ab 141b 152ab 132b 189a 13.2 <0.001
Tyrosine 113ab 94bc 100abc 74c 126a 11.1 <0.001
Citrulline 69ab 60b 70ab 73ab 81a 6.3 0.022
Ornithine 150 153 138 134 144 14.8 0.681
Urea nitrogen 2.38a 1.63bc 1.36c 1.94b 1.43c 0.173 <0.001

NCG, N-carbamylglutamate; NP, normal diet (136.4 g/kg CP); NPN, non-protein nitrogen, diammonium phosphate was used as the source of non-
protein nitrogen; LNCG, low protein diet supplemented with NCG; LNN, low protein diet supplemented with NPN and NCG; LNPN, low protein
diet supplemented with NPN; LP, low protein diet (111.4 g/kg CP); SEM, the standard error of the mean.
a− c
Means within a row without a common superscript letter differ (P < 0.05).
d
There were 6 barrows per treatment (one barrow from each of the 30 pens) in the present study.

Table 5
Effects of dietary supplementation with NPN and/or NCG on the carcass traits and meat quality of finishing pigs.
Item NP LP LNCG LNPN LNN SEM Effect (P-values)

Number of animalsd 6 6 6 6 6
Carcass traits
Carcass weight, kg 74.46 75.30 75.12 74.26 73.82 3.773 0.992
Dressing percentage, kg/100 kg BW 69.69 69.58 70.20 70.89 70.89 0.822 0.358
Tenth rib fat depth, cm 2.63ab 2.54ab 2.21b 2.86a 2.67ab 0.162 0.011
Loin-eye area, cm2 42.48 40.87 43.05 35.98 40.77 2.651 0.101
Fat-free lean, kg/100 kg CW 49.25ab 49.01ab 51.13a 46.02b 48.51ab 1.081 <0.001
Meat quality
Color L* (lightness) 50.56 52.69 50.12 51.44 53.63 2.350 0.552
a* (redness) 16.58 16.03 15.58 15.85 16.67 0.562 0.269
b* (yellowness) 3.47 3.46 2.61 3.28 3.61 0.688 0.631
pH45min 6.16 6.24 6.14 6.21 6.18 0.071 0.753
pH24h 5.43 5.52 5.51 5.48 5.51 0.080 0.782
Drip loss, g/100 g muscle 2.47 2.53 3.01 2.44 2.30 0.472 0.631

BW, body weight; CW, carcass weight; NCG, N-carbamylglutamate; NP, normal diet (136.4 g/kg CP); NPN, non-protein nitrogen, diammonium
phosphate was used as the source of non-protein nitrogen; LNCG, low protein diet supplemented with NCG; LNN, low protein diet supplemented with
NCG; LNPN, low protein diet supplemented with NPN; LP, low protein diet (111.4 g/kg CP); SEM, the standard error of the mean.
a− c
Means within a row without a common superscript letter differ (P < 0.05).
d
There were 6 barrows per treatment (one barrow from each of the 30 pens) in the present study.

condense into carbamoyl phosphate (Meijer et al., 1985). In general, NPN will form ammonia (NH3/NH+ 4 ) and be eliminated quickly
when it enters the body. Otherwise, excessive ammonia could cause toxicity to the brain and other organs of body (Dasarathy et al.,
2017). Previous studies have shown that NCG supplementation could decrease the concentrations of blood ammonia (Frank et al.,
2007; Ah et al., 2010; Chacher et al., 2014; Wang et al., 2018b). In the present study, LNN group increased the concentrations of
arginine, glycine, serine, tyrosine, and citrulline in serum of pigs compared with LP group while decreased the urea nitrogen con­
centrations in serum of pigs compared with LNPN group, suggesting that the increased ammonia by NPN supplementation could be
utilized as a nitrogen source for the endogenous synthesis of some NEAA in the presence of NCG. Moreover, it is noteworthy that
without NPN as a potential nitrogen source, adding NCG to the LP diet alone did not significantly improve the endogenous synthesis of
NEAA, as reflected in both this experiment and our previous experiment (Ye et al., 2017). These results indicated that there may be a
synergistic effect between NCG and NPN in promoting the endogenous synthesis of NEAA, which may be the main reason for the
enhanced growth performance of LNN group in the present study. However, to date, there is little knowledge about the effects of NPN

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P.L. Li et al. Animal Feed Science and Technology 273 (2021) 114753

Table 6
Effects of dietary supplementation with NPN and/or NCG on the amino acid profile (g/100 g) of M. longissimus lumborum in finishing pigs.
Item NP LP LNCG LNPN LNN SEM Effect (P-values)

Numer of animalsc 6 6 6 6 6
Arginine 1.52 1.50 1.51 1.48 1.48 0.042 0.811
Histidine 1.04 1.07 1.03 1.03 0.99 0.041 0.342
Isoleucine 1.34 1.26 1.29 1.18 1.29 0.059 0.150
Leucine 1.75 1.80 1.84 1.77 1.74 0.063 0.451
Lysine 2.09 2.04 2.03 2.02 2.00 0.041 0.422
Methionine 0.66 0.63 0.62 0.64 0.63 0.023 0.452
Phenylalanine 1.00 0.97 0.97 1.00 0.95 0.021 0.338
Threonine 1.16 1.10 1.11 1.14 1.08 0.042 0.301
Tryptophan 0.29 0.28 0.26 0.28 0.29 0.011 0.264
Valine 1.22 1.21 1.21 1.20 1.20 0.029 0.832
Alanine 1.54 1.49 1.51 1.58 1.52 0.064 0.639
Aspartate 2.29a 2.18ab 2.25ab 2.18ab 2.16b 0.043 0.011
Asparagine 0.25 0.23 0.23 0.24 0.23 0.011 0.152
Glutamate 3.34a 3.19b 3.30ab 3.31ab 3.18b 0.052 0.013
Glutamine 1.09 1.05 1.02 1.12 1.05 0.063 0.412
Glycine 1.11 1.06 1.09 1.16 1.05 0.041 0.133
Serine 0.98 0.94 0.94 0.96 0.92 0.033 0.232
Tyrosine 1.02 0.99 1.00 1.03 0.98 0.052 0.780

NCG, N-carbamylglutamate; NP, normal diet (136.4 g/kg CP); NPN, non-protein nitrogen, diammonium phosphate was used as the source of non-
protein nitrogen; LNCG, low protein diet supplemented with NCG; LNN, low protein diet supplemented with NPN and NCG; LNPN, low protein
diet supplemented with NPN; LP, low protein diet (111.4 g/kg CP); SEM, the standard error of the mean.
a− b
Means within a row without a common superscript letter differ (P < 0.05).
c
There were 6 barrows per treatment (one barrow from each of the 30 pens) in the present study.

Table 7
Effects of dietary supplementation with NPN and/or NCG on the IMF and fatty acid composition of M. longissimus lumborum in finishing pigs.
Item NP LP LNCG LNPN LNN SEM Effect (P-values)

Number of animalse 6 6 6 6 6
IMF, g/100 g muscle 2.03c 2.30bc 2.64ab 2.34bc 2.91a 0.191 <0.001
Fatty acid composition, g/100 g total fatty acids
C12:0 0.11 0.10 0.11 0.11 0.10 0.012 0.641
C14:0 1.41 1.37 1.44 1.38 1.38 0.073 0.860
C16:0 25.21b 26.33a 26.05ab 26.11ab 26.42a 0.301 0.006
C18:0 14.62 14.72 14.32 15.44 15.13 0.442 0.128
C20:0 0.25 0.23 0.22 0.27 0.26 0.020 0.259
Saturated fatty acids 42.70b 43.61ab 43.12ab 44.20a 44.11ab 0.484 0.022
C16:1 2.88 3.03 2.96 3.03 2.92 0.182 0.895
C18:1 n9 40.36b 40.60ab 40.44b 40.79ab 41.73a 0.419 0.026
C20:1 n9 0.71ab 0.74ab 0.64b 0.74ab 0.86a 0.051 0.008
Monounsaturated fatty acids 44.08b 44.46ab 44.13ab 44.65ab 45.58a 0.458 0.025
C18:2 n6 10.55a 9.96ab 10.58a 9.25bc 8.39c 0.361 <0.001
C18:3 n3 0.38 0.35 0.34 0.33 0.32 0.032 0.404
C20:4 n6 1.73a 1.18b 1.36b 1.12b 1.20b 0.101 <0.001
C20:5 n3 0.06 0.05 0.06 0.05 0.05 0.013 0.209
C22:6 n3 0.15a 0.11b 0.10b 0.11b 0.10b 0.009 0.002
Polyunsaturated fatty acids 13.22a 11.92bc 12.75ab 11.15cd 10.31d 0.392 <0.001

IMF, intramuscular fat; NCG, N-carbamylglutamate; NP, normal diet (136.4 g/kg CP); NPN, non-protein nitrogen, diammonium phosphate was used
as the source of non-protein nitrogen; LNCG, low protein diet supplemented with NCG; LNN, low protein diet supplemented with NPN and NCG;
LNPN, low protein diet supplemented with NPN; LP, low protein diet (111.4 g/kg CP); SEM, the standard error of the mean.
a− d
Means within a row without a common superscript letter differ (P < 0.05).
e
There were 6 barrows per treatment (one barrow from each of the 30 pens) in the present study.

and NCG on pig performance, and more researches needed to be conducted.

In addition, adding NPN or NCG alone to the LP diets did not affect the CATTD of nutrients while LNN group increased the CATTD
of GE, CP, EE, aNDF and ADF when compared with LP group, which may be another reason for the improved performance of pigs fed
LNN diets. The higher nutrient digestibility may due to the improvement of intestinal development in LNN group since previous studies
have found that NCG supplementation could increase the intestinal weights and morphology of pigs (Wu et al., 2010) and the relative
weight of small intestine of goat kidlets (Wang et al., 2019b). It should be pointed out that the increase of EE digestibility may be due to
the higher level of oil added in LNN group, because dietary soybean oil supplementation can improve the digestibility of nutrients (Li
and Sauer, 1994; Jørgensen and Fernández, 2000). In addition, considering that LNCG group just numerically increased the nutrient
digestibility in the present study, the role of NCG in improving nutrient digestibility may be promoted in the presence of NPN, which

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requires more researches to confirm.

Carcass and meat quality are important indicators affecting the economic benefits of pork processing industry and consumer
purchasing behavior. Among them, the carcass lean percentage is one of the main concern index of carcass quality, which is closely
related to the backfat thickness and loin-eye area (McLaren et al., 1989). In the present study, feeding NPN or NCG alone did not affect
the carcass traits or the meat quality compare to the LP group, which is consistent with the results of Wang et al. (2019a). However, Ye
et al. (2017) reported that adding NCG to LP diet significantly increased the loin eye area and tended to decrease the 10th rib fat depth.
Moreover, a previous study found that NPN supplementation (3.9 g/kg diammonium phosphate and 5.2 g/kg diammonium citrate)
tended to decrease the backfat thickness (Wehrbein et al., 1970), whereas Grimson and Bowland (1971) observed a significant
decrease in the loin area and an increased tendency for the backfat thickness of finishing pigs when diets were supplemented with
20 g/kg urea. These inconsistent results may be due to the different diet composition or dosage of NPN or NCG supplemented in
different trials. It should be noted that there are only 6 samples available per treatment for carcass and meat quality analysis in present
study, which may also be a possible reason for the difference of carcass characteristics.
The concentration of intramuscular fat is an important indicator that affecting meat quality since it is closely related to the flavor,
juiciness, tenderness and the overall acceptability of pork (Hodgson et al., 1991; Fernandez et al., 1999). The present study found that
LNN group had higher concentration of IMF than LP group and NPN group, which may due to its higher serum arginine concentration.
Because previous studies have found that dietary arginine supplementation beneficially increased IMF in finishing pigs (Ma et al.,
2010) and enhanced fat content of M. longissimus lumborum in growing-finishing pigs (Tan et al., 2009). Besides, the dietary CP level
may be another factor that affecting IMF concentration since reducing dietary CP level increased the IMF content through regulation of
the enzymes and transcription factors related to lipid metabolism (Madeira et al., 2013; Li et al., 2018), which also explained to some
extent why LNN group and LNCG group had greater IMF than NP group.
The fatty acid composition of M. longissimus lumborum in finishing pigs was analyzed in the present experiment. Compared to NP
group, LP group increased the concentration of C16:0 and decreased the concentrations of C20:4, C22:6 and PUFA, which is consistent
with the results of Teye et al. (2006) and Wood et al. (2013). Similarly, Alonso et al. (2010) reported that low protein diets decreased
the concentration of C18:2, C20:4 and PUFA in the M. longissimus lumborum of finishing pigs, and our previous study found a decrease
in the concentrations of muscular C20:4 and C22:6 in finishing pigs fed low protein diets (Ye et al., 2017). When compared with the LP
group, the combination of NPN and NCG supplementation decreased the concentrations of C18:2 and PUFA in the M. longissimus
lumborum of finishing pigs. The possible reason may be that LNN group had higher concentrations of IMF (Alonso et al., 2010; Madeira
et al., 2014). The IMF content was positively related to the SFA and MUFA proportions, and negatively related to PUFA proportions
(Ntawubizi et al., 2009). However, the research about effects of NPN and NCG on muscular fatty acid profile in finishing pigs is little at
present, further studies in this area are still needed.

5. Conclusion

The present study indicated that non-protein nitrogen or N-carbamylglutamate supplementation individually in low protein diet
did not affect the growth performance of finishing pigs, whereas non-protein nitrogen and N-carbamylglutamate supplementation in
the low protein diet increased the average daily gain of finishing pigs by 7.81 %. This is probably because their combination sup­
plementation increased the coefficients of apparent total tract digestibility of nutrients (gross energy, crude protein, ether extract,
neutral detergent fiber and acid detergent fiber) and serum amino acid concentrations (arginine, isoleucine, threonine, glycine, serine,
tyrosine and citrulline) of finishing pigs. Supplementation with both non-protein nitrogen and N-carbamylglutamate in low protein
diet did not alter the amino acid profiles but decreased the concentrations of C18:2 and polyunsaturated fatty acids, and increased the
intramuscular fat in the M. longissimus lumborum of finishing pigs. These findings suggest that combination of non-protein nitrogen
and N-carbamylglutamate supplementation in low protein diet balanced with amino acids has important complementary advantages
in pig industry for improving the growth performance and meat quality.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This study was supported by the Beijing Swine Innovation Team of Modern Agriculture Industry Technological System (Beijing,
China), and Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of China Agricultural University
(Beijing, China).

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