Effects of low-protein diet on the intestinal morphology, digestive enzyme activity, blood urea nitrogen, and gut microbiota and metabolites in weaned pigs

Abstract

This study investigated the effects of low-protein diet supplemented with Lysine (Lys), Methionine (Met), Threonine (Thr), and Tryptophan (Trp) on small intestine morphology, enzyme activity, blood urea nitrogen, and gut microbiota and metabolites in weaned piglets. Eighteen weaned pigs weighing an average of 9.57 kg received one of three treatments: a normal protein diet with 20% crude protein (CP, diet [NP]), a moderately reduced protein diet with 17% CP (MP), or a low-protein diet with 14% CP (LP). All three diets were supplemented with Lys, Met, Thr and Trp to meet essential amino acid requirements for post-weaned piglets according to the NRC (2012). Following a 45 d study period, piglets on the LP and MP diets demonstrated atrophic small intestinal morphology, with decreased villus heights and lower ratios of villus height to crypt depth (p < 0.05); pepsin activity in the stomach was also reduced in these two groups (p < 0.05). Increased plasma cholesterol and decreased blood urea nitrogen presented in the MP and LP groups compared with the NP group (p < 0.05). Overall, gastrointestinal hormones were not affected by dietary protein levels with the exception of reduced somatostatin levels in the MP and LP groups. Jejunum and colon microbiota were not affected at either the phyla or genera level in any of the diets. Colonic ammonia nitrogen concentration was reduced in MP and LP groups. Dietary protein level had no effect on short chain fatty acids or biogenic amines. Our data suggest that reducing dietary protein levels by 3% (MP) or 6% (LP) in weaned pigs has the potential to decrease nitrogen emissions and impaired digestive capacity. Therefore, dietary protein level cannot be reduced by more than 3% in consideration of maladaptive changes to small intestinal morphology and pepsin activity in weaned piglets.

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Effects of low-protein diet on the intestinal
morphology, digestive enzyme activity, blood urea
nitrogen, and gut microbiota and metabolites in
weaned pigs
Defu Yu, Weiyun Zhu & Suqin Hang
To cite this article: Defu Yu, Weiyun Zhu & Suqin Hang (2019): Effects of low-protein diet on
the intestinal morphology, digestive enzyme activity, blood urea nitrogen, and gut microbiota and
metabolites in weaned pigs, Archives of Animal Nutrition, DOI: 10.1080/1745039X.2019.1614849
To link to this article: https://doi.org/10.1080/1745039X.2019.1614849
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Effects of low-protein diet on the intestinal morphology,
digestive enzyme activity, blood urea nitrogen, and gut
microbiota and metabolites in weaned pigs
Defu Yu, Weiyun Zhu and Suqin Hang
Laboratory of Gastrointestinal Microbiology, Nanjing Agricultural University, Nanjing, China
ABSTRACT
This study investigated the effects of low-protein diet supplemen-
ted with Lysine (Lys), Methionine (Met), Threonine (Thr), and
Tryptophan (Trp) on small intestine morphology, enzyme activity,
blood urea nitrogen, and gut microbiota and metabolites in
weaned piglets. Eighteen weaned pigs weighing an average of
9.57 kg received one of three treatments: a normal protein diet
with 20% crude protein (CP, diet [NP]), a moderately reduced
protein diet with 17% CP (MP), or a low-protein diet with 14%
CP (LP). All three diets were supplemented with Lys, Met, Thr and
Trp to meet essential amino acid requirements for post-weaned
piglets according to the NRC (2012). Following a 45 d study period,
piglets on the LP and MP diets demonstrated atrophic small
intestinal morphology, with decreased villus heights and lower
ratios of villus height to crypt depth (p< 0.05); pepsin activity in
the stomach was also reduced in these two groups (p< 0.05).
Increased plasma cholesterol and decreased blood urea nitrogen
presented in the MP and LP groups compared with the NP group
(p< 0.05). Overall, gastrointestinal hormones were not affected by
dietary protein levels with the exception of reduced somatostatin
levels in the MP and LP groups. Jejunum and colon microbiota
were not affected at either the phyla or genera level in any of the
diets. Colonic ammonia nitrogen concentration was reduced in MP
and LP groups. Dietary protein level had no effect on short chain
fatty acids or biogenic amines. Our data suggest that reducing
dietary protein levels by 3% (MP) or 6% (LP) in weaned pigs has
the potential to decrease nitrogen emissions and impaired diges-
tive capacity. Therefore, dietary protein level cannot be reduced
by more than 3% in consideration of maladaptive changes to small
intestinal morphology and pepsin activity in weaned piglets.
ARTICLE HISTORY
Received 2 October 2018
Accepted 25 April 2019
KEYWORDS
Low-protein diets; weaned
pigs; intestinal morphology;
pepsin activity; blood urea
nitrogen; gut microbiota
1. Introduction
A shortage of high-quality protein sources remains a persistent problem in China.
Soybean imports reached 8.391 million tons in 2016, accounting for more than 26% of
the worldwide soybean production, and 435.04 million pigs are maintained by swine
producers at any given time. The high-protein diet administered to these animals and
resultant excretion of excess nitrogen worsens the shortage of nitrogen sources and is
CONTACT Suqin Hang suqinhang69@njau.edu.cn
ARCHIVES OF ANIMAL NUTRITION
https://doi.org/10.1080/1745039X.2019.1614849
© 2019 Informa UK Limited, trading as Taylor & Francis Group

a major contributor to environmental pollution (Portejoie et al. 2004). Therefore,
reducing the dietary protein content below that of the National Research Council
(1998) recommendations and supplementing diets with crystalline amino acids could
potentially reduce pressure on the protein ingredient supply and mitigate nitrogen
waste run-offfrom intensive pig farms.
An overarching concern is whether growth performance will be affected by reduced
dietary protein levels. Previous studies have shown that an approximate 4% reduction
of dietary crude protein (CP) level below NRC (1998) recommendations, supplemented
with (Lys, Met, Thr, Trp), did not lower growth performance in pigs at weaning,
growing, and finishing stages (Figueroa et al. 2002; Htoo et al. 2007; Zhou et al.
2015). Conversely, Nyachoti et al. (2006) found decreased growth performance in
correlation to a drop in CP, which declined from 23 to 19% or 17% despite supple-
mentation with Lys, Met, Thr and Trp. Our previous work has also shown impaired
growth performance in weaned pigs when dietary protein was reduced from 20 to 14%
even supplemented with the same four amino acids (Lys, Met, Thr and Trp) (Luo et al.
2015;Wuetal.2018). These data suggest that piglets are subject to growth retardation
when dietary protein is reduced by more than 4% (Powell et al. 2011). Recent studies
showed that branch chain amino acids (BCAA) (valine and isoleucine) were the limit-
ing factor for impairing growth performance associated with a reduction in dietary CP
(Zheng et al. 2016). Uncovering of specific mechanisms for how low-protein diets lead
to reduced growth performance, however, requires further investigation.
Growth depression in weaned piglets has been found to be associated with changes
to digestive enzyme activity and intestinal morphology (Lallès et al. 2002). The litera-
ture is inconclusive with regards to the influence of low-protein diets on villus height.
Villus height decreased in the duodenum and jejunum following weaning and in
conjunction with a reduction of dietary CP from 23.1 to 17.2%, even though diets
were supplemented with eight essential amino acids (Yu and Qiao 2008), whereas
Nyachoti et al. (2006) observed no decrease in intestinal morphology when progres-
sively reducing dietary CP. Other studies have shown that the protein diets supple-
mented with limited amino acids improved the utilisation efficiency of nitrogen without
affecting its digestibility and retention of nitrogen (Le Bellago and Noblet 2002).
Intestinal microbiota play an important role in host physiology and metabolism (Lee
and Hase 2014). Researches about the composition of microbiota in weaned piglets on
a low-protein diet had yielded inconsistent results. No effect on microbial populations
in the ileal and colon digesta was shown when CP levels were reduced from 23.0 to
17.0% (Nyachoti et al. 2006). A reduction in dietary CP level from 22.5 to 17.6% also
had no effect on short-chain fatty acids (SCFA) in weaning pigs (Opapeju et al. 2009),
although our previous study found that bacterial Shannon diversity indices and num-
bers of Firmicutes and Clostridium cluster IV were reduced in the caecal digesta, along
with lowered concentrations of acetate and branch chain fatty acids (BCFA), when CP
amounts were reduced from 20.0 to 14.0% (Luo et al. 2015). It has been well-established
that the structure of bacterial composition remains relatively unstable following wean-
ing; more studies on this question are still needed.
Previous study found that reducing dietary CP level from 20 to 14% decreased the
growth performance in weaned piglets (Luo et al. 2015;Wuetal.2018). However, the
underlying mechanism is unknown. Considering that the nutrient digestion and
2D. YU ET AL.

absorption and gut microbiota are closely associated with the pig growth, the hypoth-
esis of this study is that reducing dietary protein affects the intestinal morphology,
digestive enzymes and gut microbiota, which maybe influence the growth performance
in pigs. The aim of the study is to investigate the response of intestinal morphology,
digestive enzymes activity, gut microbiota and its metabolites to low-protein diets. It
will provide a theoretical explanation for decreased growth performance and lay
a foundation for using the low-protein diets efficiently in practice and save a large
quantity of protein resource.
2. Materials and methods
All experimental protocols were approved by the Animal Ethics Committees of the
Institute of Subtropical Agriculture and Nanjing Agricultural University.
2.1. Experimental diets
Three diets were formulated following NRC guidelines (2012)(Table 1). Differences among
the three diets were the proportion of CP therein. Dietary treatments were as follows: (1)
normal protein diet with 200 CP/kg (NP); (2) moderately reduced protein diet with 170 CP/
kg supplemented with Lys, Met, Thr, and Trp (MP); (3) low-protein diet with 140 CP/kg
supplemented with Lys, Met, Thr, and Trp (LP). Lys, Met, Thr, and Trp were provided
according to NRC recommendations (2012) for the MP and LP groups.
2.2. Pigs and housing
A total of 18 cross-bred barrows (Duroc × (Landrace × Yorkshire)), with an initial BW
of 9.57 ± 0.61 kg (mean ± SEM), were randomly assigned to three experimental diets,
each group has six replicates. Pigs were weaned at 28 d and allowed to adapt to their
new environment for 3 days before commencing their experimental diets. Pigs were
individually housed in metabolic cages equipped with a feeder and a nipple drinker, and
all pigs had ad libitum access to food and water over the 45-day trial.
2.3. Blood samples
On day 45, each of the 18 pigs was sedated with an intravenous (IV) injection of
sodium pentobarbital (50 mg/kg) the morning after an overnight fast. Blood samples
were collected by jugular venipuncture, and spun down at 3000 gat 4°C for 15 min.
Serum was decanted and immediately stored at −20°C until analysis.
2.4. Analysis of serum biochemical values and blood hormones
Serum biochemical values, including total protei
n,bloodureanitrogen,glucose,cholesterol
and triglycerides were evaluated using commercial kits according to the manufacturer’s
instructions (Nanjing Angle Gene Biotechnology, China). Blood hormones were analysed
using the commercially available ELISA kits specific for porcine tissues according to manu-
facturer’s instructions (GIP ELISA Kit [ANG-E31000P]; GLP-1 ELISA Kit [ANG-E311182P];
ARCHIVES OF ANIMAL NUTRITION 3

PYY ELISA Kit [ANG-E31368P]; SS ELISA Kit [ANG-E31118P]; CCK ELISA Kit [ANG-
E31052P]; Ghrelin ELISA Kit [ANG-E31187P]; Ins ELISA Kit [ANG-E31113P]; GH ELISA
Kit [ANG-E31119P], Nanjing Angle Gene Biotechnology, China).
2.5. Intestinal histological assessment and digestive enzyme analysis
The abdomen was exposed following jugular exsanguination. The gastrointestinal tracts
were immediately removed and rinsed with saline (0.9% NaCl). The digesta of the
stomach, jejunum, ileum and colon were separately collected and stored at −20°C until
analysis. Intestinal tissues from the duodenum, jejunum and ileum (approximately 2 cm
long, each) were obtained and stored in 10% formaldehyde solution. Villus height and
crypt depth were measured. Briefly, each tissue sample was used to prepare five slices,
and each slice was divided into three 5 µm sections, and stained in haematoxylin and
eosin to visualise intact, well-oriented crypt–villus units selected for intestinal morphol-
ogy analysis (Scion Image software, Version 4.02, 2004).
Table 1. Ingredients and nutrient composition of experimental diets.
Dietary protein level [%]
NP (20% CP) MP (17% CP) LP (14% CP)
Ingredients [%]
Corn 63.70 66.50 71.80
Soybean meal 19.80 18.80 13.40
Whey powder 4.30 4.30 4.40
Fish meal 9.00 4.00 1.50
Soya bean oil 0.80 2.60 4.10
L-Lysine 0.38 0.62 0.88
DL-Methionine 0.10 0.19 0.27
L-Threonine 0.09 0.21 0.33
L-Tryptophan 0.01 0.04 0.08
Monocalcium phosphate 0.00 0.74 1.15
Limestone 0.52 0.70 0.79
NaCl 0.30 0.30 0.30
Mineral premix* 1.00 1.00 1.00
Nutrient composition
#
[% in dry matter]
Metabolisable energy [MJ/kg] 14.54 14.55 14.56
Crude protein 20.27 17.32 14.14
Lysine 1.26 1.25 1.26
Methionine + cysteine 0.63 0.65 0.63
Threonine 0.76 0.75 0.76
Tryptophan 0.20 0.20 0.20
Arginine 1.09 0.93 0.71
Histidine 0.44 0.37 0.30
Isoleucine 0.71 0.60 0.46
Leucine 1.52 1.32 1.11
Phenylalanine 0.81 0.70 0.56
Valine 0.72 0.64 0.54
Calcium 0.70 0.71 0.70
Phosphorus 0.57 0.55 0.53
Starch 40.22 41.95 45.16
Neutral detergent fibre 8.54 8.66 8.40
Acid detergent fibre 3.29 3.30 3.05
*Premix provided per kg of complete diet: 3800 IU vitamin A, 800 IU vitamin D
3
, 9 mg vitamin E, 1 mg
vitamin K
3
, 1 mg vitamin B
1
, 2 mg vitamin B
2
, 1.2 mg vitamin B
6
, 10 µg vitamin B
12
, 10 mg nicotinic
acid, 50 µg biotin, 0.4 mg folic acid, 80 mg iron (as FeSO
4
·H
2
O), 5 mg copper as (CuSO
4
·5H
2
O), 80 mg
zinc (as ZnSO
4
·H
2
O and ZnO), 0.14 mg iodine (as KI), 0.25 mg Se (as Na
2
SeO
3
), 3 mg Mn (as MnSO
4
·H
2
O);
#
Analysed values except for digestible energy.
4D. YU ET AL.

Pepsin from the gastric juice and trypsin from the digesta of the jejunum and ileum
were analysed using commercially available pepsin and trypsin assay kits according to
manufacturer’s instructions (ANG-SH-21041 and ANG-SH-21052, Nanjing Angle Gene
Biotechnology, China).
2.6. SCFA and biogenic amines
Approximately 0.3 g digesta from each sample was mixed with 1.2 ml of distilled water.
The mixture was vortexed and centrifuged at 3000 gfor 10 min, then 0.8 ml of
supernatant was transferred into new tubes containing 0.16 ml of 25% (w/v) metapho-
sphoric acid, and stored at −20°C. After thawing, the supernatant was centrifuged at
12,000 gfor 5 min to remove particles. Concentrations of SCFA were analysed using gas
chromatography (Agilent GC-2014, Japan) with flame ionisation detector and capillary
column (Agilent Technologies Inc., USA).
High performance liquid chromatography (HPLC) was used to determine the concen-
tration of biogenic amines. Briefly, 0.5 g of digesta was mixed with 1 ml trichloroacetic acid
solution in a 2 ml centrifuge tube and centrifuged at 3000 g for 10 min. Supernatant of
samples were derived using dansyl chloride following extraction with n-hexane. A gradient
elution with two solvents was processed; solvent A consisted of HPLC-grade water, and
solvent B was acetonitrile, the flow rate for both was 1.0 ml/min. The ultraviolet detector
was set to 254 nm, and the column temperature was maintained at 30°C. For biogenic
amines analysis, each digesta was acidified with 0.2 mol/l of HCl, and concentrations were
determined by spectrophotometer (UV-2450, Shimadzu, Japan) using the method
described by Nyachoti et al. (2006).
2.7. Illumina MiSeq sequencing and bacterial data processing
The V3-V4 regions of bacterial 16S rRNA gene were amplified using a universal forward
primer, 5′-ACTCCTRCGGGAGGCAGCAG-3′, and the following reverse primer: 5′
GGACTACCVGGGTATCTAAT-3′. PCR products were detected using 2% agarose gel
electrophoresis and purified with AxyPrep DNA Purification kit (Axygen Biosciences,
Union City, USA). PCR products were visualised on agarose gels and quantitatively
determined using QuantiFluor-ST Fluoremeter (Promega, Wisconsin, USA) and
PicoGreen dsDNA Quantitation Reagent (Invitrogen, Carlsbad, USA). Purified amplicons
were pooled in equimolar amounts and paired-end sequenced (2 × 300) on an Illumina
MiSeq platform (Majorbio, Shanghai, China) according to the standard protocols.
Sequencing data were then optimised for bioinformatics analysis. Raw FASTQ files were
de-multiplexed and quality-filtered using QIIME (version 1.17) with the following criteria: (1)
300-bp reads were truncated at any site that obtained an average quality score of < 20 over
a 10-bp sliding window and truncated reads shorter than 50 bp were discarded; (2) exact
barcode matching, two nucleotide mismatch in primer matching, and reads containing
ambiguous characters were removed; and (3) only overlapping sequences longer than 10 bp
were assembled according to overlapped sequence. Reads that could not be assembled were
discarded.
Operational taxonomic units (OTU) with a 97% similarity cut-offwere clustered
using UPARSE (version 7.1), and chimeric sequences were identified and removed
ARCHIVES OF ANIMAL NUTRITION 5

using UCHIME. A rarefaction analysis based on Mothur v.1.21.1 was conducted to
reveal diversity indices, including Chao, Shannon and coverage indices.
2.8. Statistical analysis
All data were analysed using the one-way analysis of variance (ANOVA), followed by
the Student-Newman-Keuls test, using the statistical software SPSS 20.0 in Windows 10.
Data were shown as mean with total standard error of mean (SEM). Differences were
considered significant when the pvalue was less than 0.05. Bacterial community figures
(Figures 1 and 2) were generated using R software tools.
3. Results
3.1. Growth performance
The data were shown by Wu et al. (2018). Briefly, the average final weight of piglets in
group LP was significantly less than that in group NP (26.2 kg and 32.2 kg, respec-
NP MP LP
020 40 60 80 100
Crude protein level (%)
a
Others
Proteobacteria
Actinobacteria
Firmicutes
NP MP LP
Relative abundance (%)
Relative abundance (%)
020 40 60 80 100
Crude protein level (%)
b
Others
Comamonas
Mitochondria_norank
Rothia
Clostridium_sensu_stricto_1
Staphylococcus
Corynebacterium
Streptococcus
Lactobacillus
Weissella
Figure 1. Effects of dietary protein level on the composition of jejunal microbiota (with relative
abundance higher than 1%) in the weaned piglets at the phylum (a) and genus (b) levels.
NP, group with 20% CP; MP, group with 17% CP supplemented with Lys, Met, Trp and Thr; LP, group with 14% CP
supplemented with Lys, Met, Trp and Thr.
6D. YU ET AL.

tively). Compared with group NP, piglets in group LP had lower average daily feed
intake (692 g vs. 838 g) and average daily gain (370 g vs. 505 g). The feed to gain ratio
was higher in group LP than that in group NP (1.86 vs. 1.65). In group MP, growth
performance of piglets had no significant difference compared with group NP.
3.2. Small intestine morphology
Villus height was reduced in both the duodenum and jejunum as dietary CP level
decreased (p< 0.05) (Table 2). The LP diet increased crypt depth in the duodenum
(p < 0.05) and tended to increase the crypt depth in the jejunum, although not
significantly (p= 0.072). There was a significant reduction of the ratio of villus height
to crypt depth in both the duodenum and jejunum as dietary protein declined
NP MP LP
Others
Bacteroidetes
Actinobacteria
Spirochaetae
Tenericutes
Firmicutes
020 40 60 80 100
Crude protein level (%)
a
Relative abundance (%) Relative abundance (%)
020 40 60 80 100
Crude protein level (%)
MP
NP LP
bOthers
Atopobium
Olsenella
Erysipelotrichaceae_incertae_sedis
Ruminococcus
Allobaculum
Mogibacterium
Turicibacter
Christensenellaceae_uncultured
Lachnospiraceae_incertae_sedis
Treponema
Peptostreptococcaceae_incertae_sedis
Subdoligranulum
RF9_norank
Streptococcus
Blautia
Lachnospiraceae_unclassified
Ruminococcaceae_uncultured
Peptostreptococcaceae_unclassified
Lactobacillus
Clostridium_sensu_stricto_1
Figure 2. Effects of dietary protein level on the composition of colonic microbiota (with relative
abundance higher than 1%) in the weaned piglets at the phylum (a) and genus (b) levels.
NP, group with 20% CP; MP, group with 17% CP supplemented with Lys, Met, Trp and Thr; LP, group with 14% CP
supplemented with Lys, Met, Trp and Thr.
ARCHIVES OF ANIMAL NUTRITION 7

(p< 0.05). However, reduced protein did not affect villus height, crypt depth, or the
ratio of villus height to crypt depth in the ileum.
3.3. Nutrient digestibility, activity of pepsin and trypsin
The nutrient digestibility data were shown by Wu et al. (2018). Briefly, the low-protein
diet had no effect on digestive energy (DE) and dry matter (DM). Weaned pigs fed the
14% CP diet showed higher digestibility of CP, Arg, His, Ile, Lys, Met, Phe, Thr, Trp,
Val, Asp, Glu, Pro and Ser than pigs fed the 20% CP diet.
Pepsin activity in the stomach decreased with the reduction of dietary protein
content (p< 0.05) (Table 3). However, trypsin activity in the digesta of the jejunum
and ileum was not affected by changes to protein content.
3.4. Blood biochemical parameters and gastrointestinal hormones
Blood urea nitrogen was lower in pigs on lower protein diets (p<0.05) (Table 4) but
concentrations of blood cholesterol increased (p< 0.05). Other blood biochemical
indices were unaffected. The low-protein dietary had no effect on gastrointestinal
hormones other than in increase in somatostatin (SS) concentration (Table 5).
Changes to levels of dietary protein had no effect on growth hormones or insulin.
Table 2. Small intestinal morphology of piglets fed different crude protein content.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
Duodenum
Villus height [μm] 432
a
350
b
322
b
15 0.002
Crypt depth [μm] 343
a
419
ab
476
b
17 0.002
Villus:Crypt
◊
[μm/μm] 1.26
a
0.85
b
0.68
b
0.07 <0.001
Jejunum
Villus height [μm] 401
a
316
b
293
b
15 0.003
Crypt depth [μm] 254 320 325 14 0.072
Villus:Crypt [μm/μm] 1.60
a
1.01
b
0.96
b
0.09 0.002
Ileum
Villus height [μm] 260 283 258 5 0.104
Crypt depth [μm] 212 208 219 12 0.941
Villus:Crypt [μm/μm] 1.31 1.42 1.21 0.07 0.523
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP, 14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean;
◊
the ratio of
villus height to crypt depth;
a,b
significant statistical differences within row were marked by different lowercase letter
above each mean (p< 0.05).
Table 3. Pepsin and trypsin activity in the digesta of piglets.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
Stomachic pepsin [U/ml] 95.0
a
34.9
b
2.8
c
11.8 <0.001
Jejunal trypsin [U/ml] 12317 12586 12555 82 0.368
Ileal trypsin [U/ml] 6274 5487 5660 181 0.183
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP,
14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean;
a,b,c
significant
statistical differences within row were marked by different lowercase letter above each mean (p< 0.05).
8D. YU ET AL.

3.5. Microbiota in the jejunum
Average reads of 16S rRNA in the three groups ranged from 34198 to 38131, differences
that were not significant. Species richness at the taxonomic level as reflected by the
abundance-based coverage estimator and Chao index was similar across all groups. The
Shannon and Simpson diversity index did not differ among groups (Table 6).
Firmicutes are the dominant phyla in the jejunum of weaned pigs, and account for
almost 90% of the total ileal bacterial community (Figure 1). No difference was
observed about the bacterial community composition in phyla and genera (data not
shown). At the genus level, Weissella and Lactobacillus were the predominant genera,
accounting for more than 55% of the total jejunum bacterial community.
3.6. Microbiota in the colon
Average 16S rRNA gene reads in the three groups ranged from 31035 to 34382, with no
significant differences among them. Species richness at the taxonomic level as reflected
by the abundance-based coverage estimator and Chao index was similar across groups.
The Shannon and Simpson diversity index also did not differ among groups (Table 6).
The dietary treatment had no effect on the bacterial community composition in the
colon of the pigs (data not shown). Firmicutes make up more than 97% of the total
colon bacterial community (Figure 2). At the genus level, Clostridium_sensu_stricto_1,
Table 4. Effects of dietary crude protein level on blood biochemical indices in weaned piglets.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
Total protein [g/l] 58.90 55.77 57.80 1.07 0.490
Albumin [g/l] 39.40 37.02 37.84 0.64 0.296
Globulin [g/l] 19.50 18.75 19.96 0.78 0.838
Glucose [mmol/l] 6.13 5.48 5.12 0.22 0.212
Blood urea nitrogen [mmol/l] 5.28
a
3.03
b
2.24
b
0.36 <0.001
Cholesterol [mmol/l] 1.95
a
2.15
b
2.44
b
0.07 0.003
Triglyceride [mmol/l] 0.36 0.54 0.68 0.06 0.155
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP,
14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean;
a,b
significant
statistical differences within row were marked by different lowercase letter above each mean (p< 0.05).
Table 5. Gut hormone in blood serum of piglets receiving different crude protein levels.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
GIP* [ng/l] 67.89 75.51 70.80 2.73 0.545
GLP-1
◊
[pg/ml] 76.61 70.35 60.32 3.44 0.149
PYY
▲
[pg/ml] 498.78 470.74 507.37 28.10 0.871
Somatostatin [pg/ml] 130.49
a
173.79
b
162.31
b
6.57 0.010
Cholecystokinin [ng/l] 156.55 147.18 155.87 9.64 0.917
Ghrelin [ng/l] 1867.39 2209.47 2461.23 118.91 0.120
Growth hormone [ng/ml] 0.47 0.51 0.48 0.07 0.963
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP,
14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean; *GIP, glucose-
dependent insulinotropic polypeptide;
◊
GLP-1, glucagon-like peptide-1;
▲
PYY, peptide tyrosine;
a,b
significant statis-
tical differences within row were marked by different lowercase letters above each mean (p< 0.05).
ARCHIVES OF ANIMAL NUTRITION 9

Lactobacillus, and Peptostreptococcaceae_unclassified, were the predominant genera,
accounting for more than 50% of the total colon bacterial community.
3.7. SCFA, biogenic amines and ammonia in the jejunum and colon
Few SCFA were found in the digesta of the jejunum, and valerate (pentanoate) decreased
correlatively with the reduction of CP in the diet (P< 0.05). In the colon, acetate and
propionate were the dominant SCFA, with no differences therein among groups. Colonic
isobutyrate tended to decrease with the decrease in protein in the diets, although this was
not found to be statistically significant (p=0.057,Table 7). No difference was observed in
the concentration of biogenic amines in the colon among the treatment and control
groups. In the jejunum, tryptamine decreased when the protein level was reduced by 6%
(Table 8). Colonic ammonia nitrogen was reduced when weaned pigs were fed low-protein
diet, although this was not found to be the case in the jejunum (Figure 3)
4. Discussion
Nitrogen runofffrom intensive pig operations has a negative impact on the envir-
onment, contributing to acidification and eutrophication of ecosystems as well as
odour emissions. One approach to decreasing nitrogen emission is to reduce dietary
CP and supplement the feed with essential amino acids. However, previous studies
have shown that low dietary protein impaired growth performance (Wu et al. 2018).
In this study, gastrointestinal digestive function as evaluated by intestinal morphol-
ogy, digestive enzyme activity, and microbiota in the small intestine and colon, was
investigated to uncover the mechanisms of reduced pig growth performance on low-
protein diets.
Table 6. Overview of the sequencing data and alpha-diversity of the microbiota in digesta.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
Jejunum
Reads 37401 34198 38131 1201 0.387
OTU
§
249 272 222 24 0.735
ACE
$
339 351 343 23 0.980
Chao 327 333 307 25 0.919
Coverage 0.998 0.998 0.998 0.0001 0.725
Shannon 2.31 2.47 1.79 0.18 0.273
Simpson 0.27 0.24 0.32 0.04 0.687
Colon
Reads 33354 31035 34382 861 0.282
OTU 382 365 376 14 0.898
ACE 479 439 452 13 0.449
Chao 487 450 455 15 0.560
Coverage 0.997 0.997 0.997 0.0001 0.577
Shannon 2.93 2.89 2.87 0.2 0.993
Simpson 0.22 0.21 0.2 0.04 0.956
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP, 14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean;
§
OTU,
operational taxonomic units;
$
ACE, abundance-based coverage estimator.
10 D. YU ET AL.

4.1. Intestinal morphology
The decline of the villus height and a deeper crypt may suggest a faster turnover of new villus
cells leading to poor nutrient absorption (Xia et al. 2005). Although low-protein diets have
been shown to improve the ileum morphology of weaned piglets under enterotoxigenic E. coli
(ETEC) challenge (Opapeju et al. 2009), generally, villus height in weaned piglets was not
affect by the reduction of dietary CP from 21 to 17% and supplemented with Lys, Met, Thr,
Trp, Ile and Val (Opapeju et al. 2008). Yue and Qiao (2008) also supplemented eight essential
amino acids to a low-protein diets with CP reduction from 23.1 to 18.9% and did not note
villus atrophy, however further reduction to 17.2% was associated with impaired villus height
in both the duodenum and jejunum. Moreover, villi height was impaired in ileum when CP
level was reduced both in growing pigs (reduced from 18 to 12% CP) and adult pigs (CP
Table 7. The concentration of short chain fatty acids (SCFA) in the digesta of piglets.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
Jejunum
Acetate [µmol/g] 1.17 0.83 0.95 0.08 0.291
Isobutyrate [µmol/g] 1.75 2.55 3.23 0.55 0.641
Butyrate [µmol/g] 1.92 1.28 0.81 0.72 0.808
Valerate [µmol/g] 1.46 0.82 0.43 0.22 0.110
Total SCFA [µmol/g] 4.86 2.94 4.00 0.66 0.551
Colon
Acetate [µmol/g] 48.10 37.17 41.65 2.76 0.281
Propionate [µmol/g] 10.92 10.94 11.50 0.76 0.947
Isobutyrate [µmol/g] 9.26 5.78 7.15 0.63 0.063
Butyrate [µmol/g] 3.50 3.19 3.91 0.33 0.694
Isovalerate [µmol/g] 5.72 4.45 5.00 0.35 0.352
Valerate [µmol/g] 3.93 3.20 4.10 0.34 0.545
Total SCFA [µmol/g] 81.43 64.72 73.31 4.15 0.274
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP,
14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean.
Table 8. The concentration of biogenic amine in the digesta of piglets.
Dietary protein level [%]
NP
#
MP
‡
LP
†
SEM
♦
p-Value
Jejunum
Methylamine [µmol/g] 0.17 0.95 0.33 0.12 0.060
Tryptamine [µmol/g] 0.18
b
0.14
ab
0.06
a
0.02 0.041
Putrescine [µmol/g] 0.37 0.36 0.33 0.04 0.933
Cadaverine [µmol/g] 0.25 0.29 0.20 0.04 0.662
Tyramine [µmol/g] 0.05 0.04 0.04 0.00 0.461
Spermidine [µmol/g] 0.13 0.09 0.09 0.02 0.587
Spermine [µmol/g] 0.08 0.09 0.11 0.01 0.471
Colon
Methylamine [µmol/g] 0.62 0.34 0.36 0.10 0.483
Tryptamine [µmol/g] 0.11 0.12 0.16 0.02 0.320
Putrescine [µmol/g] 0.75 0.74 0.75 0.05 0.998
Cadaverine [µmol/g] 0.27 0.31 0.31 0.03 0.834
Tyramine [µmol/g] 0.19 0.19 0.11 0.05 0.777
Spermidine [µmol/g] 0.20 0.51 0.33 0.06 0.105
Spermine [µmol/g] 0.10 0.13 0.08 0.02 0.554
#
NP, 20% crude protein according to NRC (2012);
‡
MP, 17% crude protein supplemented with Lys, Met, Trp and Thr;
†
LP,
14% crude protein supplemented with Lys, Met, Trp and Thr;
♦
SEM, standard error of the mean;
a,b
significant
statistical differences within row were marked by different lowercase letter above each mean (p< 0.05).
ARCHIVES OF ANIMAL NUTRITION 11

reduced from 16 to 10%) (Chen et al. 2018;Fanetal.2017). Probably, pigs are unable to
maintain the gut architecture of intestinal epithelium due to protein deficiency. Fan et al.
(2017) revealed that the expression of Lgr5 and Bmil, the markers of intestinal stem cells
(ISC), was decreased significantly when the dietary protein level was reduced, indicating
proliferation of ISC was suppressed and could not support villi growth. Therefore, protein
deficiency results in the impaired intestinal morphology through inhibiting the growth of
ISC. In alignment with these data, we also observed lower villus height and deeper crypt depth
in the duodenums and jejunums in weaned pigs fed 17 or 14% CP diets supplemented with
Lys, Met, Thr, Trp, which partly explained the reduced growth performance showed in
previous study (Luo et al. 2015;Wuetal.2018). On the other hand, Powell et al. (2011)
reported that the supplementation of valine and isoleucine to the low-protein diet (reduced
from 18% to 13% CP supplemented with Lys, Met, Thr, Trp) restored average daily gain in
growing pigs. Similar results were reported by Zheng et al. (2016), that supplementing BCAA
to LP diets restored growth performance of weanling piglets by increasing the feed intake and
skeletal muscle growth in piglets, implying that BCAA may be the next-limiting amino acid
(AA) in low-protein diets. Thus, we recommend that BCAA should be added to the diets as
the CP level reduced. Considering the different condition in farms, more studies need to be
conducted in different farms and large number of pigs should be also involved in.
4.2. Activity of pepsin and trypsin
Growth depends on digestive capacity in all animals, digestive processes include glucose
and amino acid utilisation, intracellular protein turnover, fat deposition, and regulation
therein by hormones. Efficient digestive enzymes are required to degrade macronutrients in
0
2
4
6
8
10
NP
MP
LP
a
b
b
Jejunum Colon
HNfonoitirtnecnoC 3]g/Mm[N-
Figure 3. The concentration of ammonia nitrogen in jejunum and colon of weaned pigs fed different
dietary protein level.
Bars represent means ± standard error; Significant statistical differences were marked by different lowercase letter (a or
b) above each bar (p-value < 0.05); NP, group with 20% crude protein; MP, group with 17% crude protein
supplemented with Lys, Met, Trp and Thr; LP, group with 14% crude protein supplemented with Lys, Met, Trp and Thr.
12 D. YU ET AL.

the diet into small molecules that can be easily absorbed. In growing and finishing pigs,
mRNA expressions of chymotrypsin and trypsinogen were not affected when dietary
protein level was reduced from 18 to 16%, but further reduction to 12% down-regulated
genetic expression of trypsinogen and up-regulated genetic expression of chymotrypsin
(He et al. 2016). Of importance, expression of digestive enzymes is subject to complex
regulation at both the transcriptional and translational levels. In this study, trypsin activity
in the digesta of the duodenum and jejunum were unaffected, which could explain the
improved nitrogen utilisation in pigs fed the MP and LPdiet. Pepsin activity in the stomach
digesta was reduced in the LP group, which may due to the factor of consuming less
protein. In addition, SS has been shown to inhibit gastric acid and pepsin secretion
(Mogard et al. 1985), and we did observe reduced amounts of SS along with decreased
pepsin activity in the LP and MP groups.
Combination with the results of digestibility shown by Wu et al. (2018), reducing the
dietary protein from 20 to 17% maintained the growth performance, partly due to the
improved digestibility of CP. When CP level was further reduced to 14%, nutrient
digestibility was also improved. However, the digestive enzymes activity and villus
height were remarkably suppressed. When CP was reduced from 20 to 17% or 14%,
low-protein diets contain more crystalline amino acids and less intact protein, which do
not need high enzymes activity to degrade intact protein. Although the digestibility of
CP was increased in low-protein diets group, the growth performance was negatively
affected. The explanation of low growth performance for pigs under condition of low
protein level (14% CP) may result from low feed intake. However, further studies are
required to determine the relationship between changes to digestive enzyme activity
and a low CP diet in pigs.
4.3. Blood urea nitrogen and cholesterol
High-protein diets are used in traditional corn-soybean meal to satisfy the requirement
of lysine because lysine is the first limiting AA. Consequently, high-protein diets led to
the overabundance of other AA, which decreased the efficiency of nitrogen utilisation.
Kohn et al. (2005) found that concentration of blood urea nitrogen was highly corre-
lated with urinary nitrogen excretion rate, and suggesting that blood urea nitrogen
could be used to predict nitrogen excretion and efficiency of nitrogen utilisation in pigs.
Blood urea nitrogen concentration and ammonia in faecal samples decreased alongside
a reduction of dietary CP levels, and were also dependent upon a balance of amino
acids (Figueroa et al. 2002; Nyachoti et al. 2006Heo et al. 2008). Therefore, in current
study, piglets were provided low-protein diets supplemented with four essential amino
acids (Lys, Met, Thr and Trp) and blood urea nitrogen was used as an indicator of
nitrogen utilisation. LP or MP diet decreased concentration of blood urea nitrogen and
ammonia in colon, indicating that nitrogen excretion was reduced and nitrogen utilisa-
tion was elevated. This result is consistent with Lordelo et al. (2008) who revealed that
nitrogen excretion from faeces and urine was reduced in piglets fed 17% CP diets
compared with 20% CP diet supplemented with Lys, Met, Thr and Trp.
Previous research revealed that pigs fed low-protein diets presented a more fat
carcass compared with high-protein diets (Morazán et al. 2015), which might result
from the ideal protein ratio in low-protein diet that would reduce energy needed for
ARCHIVES OF ANIMAL NUTRITION 13

N excretion leaving more energy to be deposited in adipose tissue (Smith et al. 1999).
Similarly, an increase of plasma cholesterol was observed and plasma triglyceride
tended to be elevated in pigs fed LP or MP diet in the present study. These results
are in agreement with the findings of Jiang et al. (2017), who observed that plasma
cholesterol in Pekin ducks was increased when dietary protein level was reduced from
22 to 16%. Whereas Nukreaw and Bunchasak (2015) found that triglyceride instead of
cholesterol in blood was significantly increased in low-protein diet supplemented with
Met and Lys in broiler chicks. Further researches on the underlying mechanisms of how
low-protein diets influence the lipid metabolism need to be conducted.
4.4. Microbiota in digesta
The pigs’gastrointestinal tract harbours a substantial number of indigenous bacteria,
mainly Lactobacillus spp. in the stomach (Conway 1994) and small intestine (Jensen and
Jørgensen 1994). Lactobacillus and Weissella were the predominant bacterial genera in
the jejunum, and their proliferation can be enhanced by fermentable carbohydrates
such as different types of resistant starch, non-starch polysaccharides (NSP), and non-
digestible oligosaccharides in the small intestine (Bikker et al. 2006). Reducing the
amount of dietary protein has been viewed as an alternative option to avoid excessive
protein fermentation in the large intestine of the pig (Heo et al. 2008). Peng et al. (2017)
reported that reduction of CP level from 20.0 to 15.3% increased bacterial diversity in
colon digesta and colon mucosa, which may decrease the risk of intestinal infection
(Manichanh et al. 2006). In this study, however, even when dietary CP levels dropped to
14%, the colonic bacterial community, SCFA and biogenic amines in jejunum and
colon were not changed in weaned piglets except for reduced amount of ammonia. The
results are different from that revealed by Chen et al. (2018), who reported that the
concentration of acetic acid, cadaverine and spermidine were decreased by low-protein
diet (12% CP) in growing pigs. The other study also demonstrated that the intestinal
concentrations of SCFA and biogenic amines were lower for growing pigs fed a low
dietary protein (10% CP) compared to that pigs fed 16% (Fan et al. 2017). Ileal
digestibility of CP was increased in group LP while the digestibility of dry matter
(DM) in group LP had no difference from that in group NP (Wu et al. 2018), which
probably led to the point that less CP content but no significantly different DM
amounts reached the colon among groups. As no indigestible fibre was added to any
of the diets, there was little fermentable carbohydrate to affect the colonic bacterial
community. Considering that the structure of bacterial composition of weaned pigs
remains relatively unstable, due to the combination of multiple factors such as chemical
composition of the diet, stress resulting from the weaning process, and other physio-
logical factors (Kim and Isaacson 2015), more replicates in each group and sampling at
different time points should be concerned for the dynamic changes of microbiota in the
future experiments.
Fermentation of protein often coincides with the growth of potential pathogens, such
as Bacteroides and Clostridium species, thereby increasing the risk of infectious diseases
(Macfarlane and Macfarlane 1995). Bacteria belonging to the Clostridium family play
an important role in amino acid utilisation in animals (Neis et al. 2015). In this study,
Clostridium_sensu_stricto_1 was the predominant genera found in the colons of
14 D. YU ET AL.

weaned pigs, and its proportion tended to be decreased when the dietary CP level
reduced, probably due to the higher ileal digestibility of CP in low-protein diet group
(Fan et al. 2017; Wu et al. 2018), which resulted in the less protein to reach colon lumen
for fermentation. Moreover, members of Clostridium were reported to be positively
associated with necrotising enterocolitis (Brower-Sinning et al. 2014). The 6% reduction
of dietary CP had greater proportion of these bacteria than 20% CP group in the
current study, indicating pigs with low-protein diet may be more susceptible to infec-
tion of necrotising enterocolitis. Likewise, the genus of Lactobacillus is a member of the
lactic acid bacterial group, which is believed to promote health in man and animals
(Kleerebezem et al. 2003). The proportion of Lactobacillus tended to increase without
a concomitant difference in the colon in this study, which was inconsistent with the
results showed by Zhou et al. (2015) that the abundance of Lactobacillus in the colon
was not affected and decreased in the cecum by reducing dietary CP. The genus of
Weissella also belongs to lactic acid bacteria and Weissella cibaria possesses the ability
to inhibit volatile sulphur compounds production under both in vitro and in vivo
conditions (Kang et al. 2010), indicating that Weissella has potential benefits for host.
Peptostreptococcaceae may help maintain gut homeostasis, as its proportion is higher
in the gut microbiota of health animals than that on patients with intestinal dysbiosis
(Leng et al. 2016). Taken together, the beneficial bacteria have a trend to occupy the
main position, especially in jejunum when pigs were fed low-protein diets supplemen-
ted with Lys, Met, Thr and Trp.
Isobutyrate and isovalerate, also known as BCFA, come from deamination of
BCCA (valine and leucine) by bacteria. Thus, BCFA are considered markers of
protein fermentation (Neis et al. 2015). In the present study, the isobutyrate in
colon tended to be decreased along with the reduced CP. It may imply that bacterial
fermentation utilising BCCA (valine and leucine) as the substrate was suppressed by
CP reduction. On the other hand, SCFA have profound effects on metabolism and
gut health. Acetate and propionate are energy substrates for peripheral tissues, and
butyrate is preferentially used as an energy source by colonic epithelial cells
(Tremaroli and Bäckhed 2012). It has been proved by previous researchers
(Bäckhed et al. 2007; Donohoe et al. 2011), who revealed that the colonic epithelial
cells and liver of germ-free mice are severely energy-deprived and differs consider-
ably from that in microbiota-colonised mice. Possibly, the increased influx of SCFA
into the liver of colonised mice led to acetate and propionate used as substrates for
lipogenesis and gluconeogenesis by the liver. SCFA also affect proliferation, differ-
entiation and modulation of gene expression in mammalian colonic epithelial cells.
These effects have been mainly attributed to butyrate acting as a potent histone
deacetylase inhibitor (Davie 2003). In addition, SCFA can regulate gene expression
by binding to the G-protein-coupled receptors in gut epithelial cells, and then
suppress inflammation in immune cells (Maslowski et al., 2009,Sinaetal.2009),
and modulate secretion of GLP-1 by enteroendocrine L-cells in the distal small
intestine and colon (Tolhurst et al. 2012). In the present study, the total SCFA
tended to be lower in the low-protein diets, suggesting that a reduction of the energy
was produced by bacteria fermentation for host and may be insufficient to contribute
to the growth of epithelial cell.
ARCHIVES OF ANIMAL NUTRITION 15

5. Conclusion
Based on NRC guidelines (2012), 17 or 14% CP diets supplemented with four
essential AA negatively affected small intestinal morphology and decreased pepsin
activity in weaned piglets. Further, decreased blood urea nitrogen and ammonia in
the colons indicated that excretion of nitrogen was reduced, and the colon micro-
environment was not compromised by these low-protein diets. Combined with the
impaired growth performance of weaned piglets fed 14% CP diets showed by Wu
et al. (2018), reducing the dietary CP level by 3% supplemented with Lys, Met, Thr,
Trp for weaned piglets is suggested. More research should be focused on the changes
in digestion and absorption capacity in small intestinal when pigs are fed low-
protein diets. Notably, not only Lys, Met, Thr, Trp but also BCAA may contribute
to an ideal amino acid pattern for piglets.
Acknowledgments
The authors appreciate the Institute of Subtropical Agriculture and The Chinese Academy of
Science, for animal feeding and assistance during sampling.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Key Basic Research Programme of China
[2013CB127301].
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