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Citation: Yang, W.; Jiang, F.; Yu, B.;
Huang, Z.; Luo, Y.; Wu, A.; Zheng, P.;
Mao, X.; Yu, J.; Luo, J.; et al. Effect of
Different Dietary Lipid Sources on
Growth Performance, Nutrient
Digestibility, and Intestinal Health in
Weaned Pigs. Animals 2023,13, 3006.
https://doi.org/10.3390/ani13193006
Academic Editor: Jin-Ho Cho
Received: 21 August 2023
Revised: 19 September 2023
Accepted: 22 September 2023
Published: 24 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
animals
Article
Effect of Different Dietary Lipid Sources on Growth
Performance, Nutrient Digestibility, and Intestinal Health in
Weaned Pigs
Wenjuan Yang 1,2, Fei Jiang 3, Bing Yu 1,2, Zhiqing Huang 1,2 , Yuheng Luo 1,2, Aimin Wu 1,2, Ping Zheng 1,2,
Xiangbing Mao 1,2 , Jie Yu 1,2, Junqiu Luo 1,2 , Hui Yan 1,2 and Jun He 1,2, *
1Institute of Animal Nutrition, Sichuan Agricultural University, Chengdu 611130, China;
muyao0462@163.com (W.Y.); ybingtian@yahoo.com.cn (B.Y.); zqhuang@sicau.edu.cn (Z.H.);
luoluo212@126.com (Y.L.); wuaimin0608@163.com (A.W.); zpind05@163.com (P.Z.);
acatmxb2003@163.com (X.M.); jerryyujie@163.com (J.Y.); junqluo2018@tom.com (J.L.);
yan.hui@sicau.edu.cn (H.Y.)
2Key Laboratory of Animal Disease-Resistant Nutrition, Chengdu 611130, China
3Singao Agribusiness Development Co., Ltd., Longyan 361000, China; jiangfeifeijiang@163.com
*Correspondence: hejun8067@163.com
Simple Summary:
Lipids are an ideal source of energy for piglets. Several studies have shown
that dietary oil supplementation can improve the growth performance of weaned piglets. This
experiment investigated the effects of different fat sources on the growth performance and intestinal
health of weaned piglets, and compared with the conventional use of soybean oil, pigs treated with
fish–palm–rice oil mixture (FPRO) and coconut–palm–rice oil mixture (CPRO) showed improvements
in digestibility and intestinal epithelial function, and a reduction in the production of inflammatory
cytokines. The results of this study can provide the theoretical basis for the rational selection of lipid
sources in weaned piglets and other mammals.
Abstract:
To investigate the effects of lipid sources on growth performance and intestinal health,
72 weaned pigs
were randomly allocated to three treatments. Pigs were fed with a corn–soybean
meal diet containing 2% soybean oil (SO), or fish–palm–rice oil mixture (FPRO), or coconut–palm–rice
oil mixture (CPRO). The trial lasted for 28 days; blood and intestinal tissue samples were collected.
The results showed that the crude fat digestibility of the FPRO group was higher than that of the SO
and CPRO groups (p< 0.05). The FPRO group also had higher digestibility of dry matter, ash, and
gross energy than the SO group (p< 0.05); compared to the SO group, the serum interlukin-6 (IL-6)
concentration was decreased. Interestingly, the FPRO and CPRO groups had higher villus height
than the SO group in the jejunum and ileum, respectively (p< 0.05). Moreover, the FPRO group had
higher Lactobacillus abundance than the SO group in the colon and cecum (p< 0.05). Importantly, the
expression levels of tight junction protein ZO-1, Claudin-1, and Occludin in the duodenal and ileal
mucosa were higher in the FPRO group than in the SO and CPRO groups (p< 0.05). The expression
levels of nutrient transporters such as the CAT-1, PepT1, FATP1, and SGLT1 were higher in the FPRO
group than in the SO group (p< 0.05). The improved digestibility and intestinal epithelium functions,
as well as the reduced inflammatory cytokines, in the FPRO and CPRO group suggest that a mixed
lipid source such as the FPRO deserves further attention.
Keywords: lipid source; growth performance; intestinal health; weaned pigs
1. Introduction
The intestinal mucosa consists of a simple columnar layer of epithelial cells and the
lamina propria and muscular mucosa beneath it, with epithelial cells on the mucosal surface
creating a barrier between the sometimes hostile external and internal
environments [1,2]
.
Animals 2023,13, 3006. https://doi.org/10.3390/ani13193006 https://www.mdpi.com/journal/animals
Animals 2023,13, 3006 2 of 14
The largest exchange surface between the body and the external environment is the in-
testinal epithelial barrier (IEB), whose permeability plays a central role in nutrient intake
and body fluid regulation and controlling the infection of pathogens [
3
]. However, var-
ious factors, such as weaning stress, oxidative stress, and bacterial infection, can impair
the IEB by inducing overproduction of pro-inflammatory cytokines and reactive oxygen
species (ROS), which subsequently leads to decreases in nutrient digestion and absorption,
systemic inflammatory response, and even the death of piglets [4–7].
Lipids can serve as an ideal energy source for piglets due to its high energy concen-
tration (2.25 times more than carbohydrates and proteins), and have been usually added
to the diet to solve the problem of insufficient energy intake during the weaning stage of
piglets [
8
]. A number of studies indicated that dietary oil supplementation can improve
the growth performance of weaned piglets. For instance, dietary lipid supplementation
significantly increased ADG and G:F of piglets from the second week to the fifth week
post-weaning, and significantly increased ADG and G:F of the piglets during the first five
weeks post-weaning [
9
]. Moreover, dietary lard supplementation significantly increased the
G:F of piglets from the second week to the third week and the third week post-weaning [
10
].
In addition to acting as an energy source, the lipids can also provide a series of essential
fatty acids (EFAs), such as linoleic acid and linolenic acid. Previous studies indicated that
the EFAs can inhibit the overproduction of intestinal inflammatory mediators, especially
various pro-inflammatory cytokines [
11
,
12
]. For instance,
in vitro
studies indicated that
Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) can inhibit the production
of IL-1
β
and tumor necrosis factor (TNF)-
α
by monocytes [
13
], and inhibit the production
of IL-6 and IL-8 by venous endothelial cells [
14
,
15
]. Moreover, dietary supplementation of
2% Conjugated linoleic acid (CLA) in second-trimester sows significantly reduced intestinal
inflammation, and increased the serum concentrations of immunoglobulin in neonatal pigs
upon enterotoxigenic Escherichia coli challenge [16].
A previous study indicated that the effect of dietary lipids supplementation on animals
may vary widely depending on their sources or chemical structures [
17
]. For instance, di-
etary supplementation of fish oil (one of the major sources of EPA) significantly reduced the
production of inflammatory cytokines such as the TNF-
α
, IL-1
β
, and IL-6 by macrophages
in rodents [
18
,
19
]. Rice oil contains a large number of compounds with antioxidant proper-
ties, such as glutamine, tocopherol, and tocotrienol [
20
]. Previous studies have shown that
dietary supplementation of rice oil can improve the growth performance of broilers [
21
,
22
]
and reduce the serum cholesterol concentration [
23
]. Compared to long-chain fatty acids
(LCFAs), medium-chain fatty acids (MCFAs) are more efficiently digested and absorbed
in the small intestine of piglets [
24
]. Coconut oil is a critical source of the MCFAs, and
a previous study indicated that the ADG of the weaned pigs during the first four weeks
post-weaning was higher in the coconut oil group than in the butter and corn oil group (the
weaner diet contains 8% oil) [
25
]. Moreover, both the ADG and ADFI of the weaned pigs
were higher in the coconut oil group than in the soybean oil group during the three to four
weeks after weaning [9].
Although, these studies indicated a beneficial effect of different lipid sources on
the weaned pigs, the combined effects of different lipid sources are just beginning to
be explored. In the present study, we investigated the effects of different lipid sources
on growth performance and intestinal health in weaned piglets. Pigs were fed with
the corn–soybean meal diet containing 2% SO or oil mixtures (FPRO and CPRO). We
observed improved digestibility and intestinal epithelium functions, as well as a reduced
production of inflammatory cytokines in the FPRO and CPRO group, which may offer a
theoretical basis for rational selection of lipid sources for the weaned pigs as well as other
mammalian animals.
Animals 2023,13, 3006 3 of 14
2. Materials and Methods
2.1. Experimental Design and Diets
A total of 72 healthy weaned pigs (average 7.80
±
0.11 kg) with similar body conditions
and breeds were randomly divided into three treatment groups with eight replicates per
treatment, and three piglets housed in a pen. Pigs were fed corn- and soybean-meal-based
diets containing 2% soybean oil (SO), fish–palm–rice oil mixture (FPRO, consisting of 10%
fish oil, 50% palm oil, and 40% rice oil), or coconut–palm–rice oil mixture (CPRO, consisting
of 5% coconut oil, 80% palm oil, and 15% rice oil), respectively. The composition and
nutrition level of the basic diet was prepared in reference to the NRC (2012) pig feeding
standard (Table 1) [
26
]. The basal diet was supplemented with 2% soybean oil and the FPRO
and CPRO groups were supplemented with 2% FPRO and CPRO, respectively. The feeding
management and immunization of the experimental group were carried out according
to the field regulations. The pre-feeding period lasted for 3 days. The experiment lasted
28 days. Pigs were supplied with feed and fresh water freely and room temperature was
controlled between 25~28
◦
C, relative humidity 65%
±
5% [
27
]. On days 0 and 29 of the
experimental period, the feed volume and weight of piglets were recorded to calculate the
average daily feed volume (ADFI), average daily gain (ADG), and feed conversion rate
(FCR) for the entire experimental period.
Table 1. Formulated diet and nutrient compositions.
Ingredients % Nutrient Level Contents
corn [Grade 8.7%] 39.56 Digestible energy of pigMC/kg 3.5
Hulled barley 8 Crude protein % 17.77
Broken rice 15 Calcium % 1.19
Wheat bran 3.5 Total phosphorus % 1.11
Soybean meal 22.6 Available phosphorus % 0.54
Secondary powder 15 digestible lysine % 1.36
Oil 22 digestible methionine % 0.32
Lysine 0.61 digestible cystine % 0.57
DL Methionine 0.08 digestible threonine % 0.77
L-threonine 0.25
L-Tryptophan 0.1
Choline chloride 50% 0.1
Vitamin premix 30.05
Mineral premix 40.25
NaCl 0.3
Limestone 0.9
Calcium hydrogen phosphate 0.9
Complex enzyme 0.05
Pig phytase (5000) 0.05
Compound acidifier 0.7
Total 100
1
Also known as black flour, yellow flour, lower- or third-class flour, it is a byproduct of wheat processing mainly
composed of finely ground bran and some wheat endosperm. It is one of the byproducts obtained from grinding
various types of flour using wheat grains as raw materials.
2
The oil added in the SO group is 2% soybean oil; the
oil added in FPRO group is 2% mixed oil 1 (palm oil:rice oil:fish oil = 5:4:1); the oil added in CPRO group is 2%
mixed oil 2 (palm oil:rice oil:coconut oil = 16:3:1).
3
The vitamin premix provided the following per kg of diet:
9000 IU of VA, 3000 IU of VD3, 20 IU of VE, 3 mg of VK3, 1.5 mg of VB1, 4 mg of VB2, 3 mg of VB6, 0.02 mg of
VB12, 30 mg of niacin, 15 mg of pantothenic acid, 0.75 mg of folic acid, and 0.1 mg of biotin.
4
The mineral premix
provided the following per kg of diet: 100 mg Fe, 6 mg Cu, 100 mg Zn, 4 mg Mn, 0.30 mg I, 0.3 mg Se.
2.2. Sample Collection
Feed samples were collected at the beginning of the experiment and stored at
−20 ◦C
for nutritional analysis. Fresh fecal samples were collected immediately after excretion
on days 25–28 of the experiment, weighed, and 10 mL 10% H
2
SO
4
solution was added to
every 100 g of fresh fecal matter [
28
]. Feed and fecal samples were dried for 2 days at 65
◦
C,
ground through a one-mesh sieve, and then stored at
−
20
◦
C until nutrient digestibility
Animals 2023,13, 3006 4 of 14
analysis. On the morning of the 29
th
day, we selected one piglet from each cage that is
closest to the average weight for blood collection and slaughter, and collected 15 mL of
venous blood. After 30 min, the blood was centrifuged at 3500 rpm and 4
◦
C for 15 min to
prepare the serum. After centrifugation, the serum was packed separately (1.5 mL EP tube,
300
µ
L/tube) and put it on ice. All blood samples were repackaged and then transferred to
−
20
◦
C refrigerator for storage. After slaughter, about 2 cm of the middle segment of the
duodenum, jejunum, and ileum were taken and rinsed gently with precooled normal saline
to remove the contents, and were then immersed in 4% paraformaldehyde for fixation
and preservation, which was used to determine the histomorphology [
29
]. Next, another
segment of the duodenum, jejunum, and ileum was cut open with a scissor, then the
contents of the intestine were washed with normal saline and the water was absorbed
with filter paper. The intestinal mucosa samples were gently scraped and immediately
frozen with liquid nitrogen, and then stored at
−
80
◦
C. The cecum and colon chyme were
squeezed into a paper cup and quickly collected with the corresponding sampling spoon.
The chyme was put into the corresponding cryopreservation tube (sterilized) [
30
]. Each
kind of intestine was put into four tubes, wrapped with tin foil, and quickly put into liquid
nitrogen for preservation.
2.3. Apparent Total Tract Nutrient Digestibility Analysis
Using Acid insoluble ash (AIA) as an endogenous indicator, the nutrient digestibility
levels of dried, ground, and fecal samples were analyzed. The contents of dry matter (DM),
crude protein (CP), crude fat (EE), and ash were determined according to AOAC [
31
]. The
energy (GE) was measured directly by a bomb calorimeter.
2.4. Serum Biochemical Indicators
Immunoglobulin (IgA, IgG, IgM), glycolipid substitute (triglyceride, cholesterol, low-
density lipoprotein, blood glucose, etc.), inflammatory factor (IL-6, TNF-
α
, IL-1
β
), antioxi-
dant indexes (T-AOC, CAT, SOD, GSH-PX, MDA), hormone and growth factors (insulin,
IGF-1) were tested in special kits built in Nanjing. The specific operation was carried out
according to the kit instructions.
2.5. Intestinal Morphology
The intestinal segments fixed with 4% paraformaldehyde were dehydrated through a
graded series of ethanol and then embedded in paraffin [
32
]. The slices (2
µ
m) were stained
with hematoxylin and eosin, observed with Leica DM1000 biological microscope under
100 times of field of vision, selected 10 appropriate fields of vision (complete villi) and
photographed, and then measured the villus height and corresponding crypt depth of a
single villus [33].
2.6. Intestinal Disaccharidase Content
The frozen duodenum, jejunum, and ileal mucosa were homogenized with a ratio of
1:9 (w/v) frozen normal saline for 15 min. Homogenates were centrifuged at 3500
×
gat
4
◦
C for 15 min, and enzyme activities were measured in the supernatant. The content of
disaccharidase (sucrase, lactase, and maltase) in the supernatant was determined with the
kit of Nanjing Jiancheng Bioengineering Research Institute.
2.7. Intestinal Flora
We weighed 0.2 g caecum and colon thawed chyme, and used EZNA
®
Stool DNA
kit (Omega Bio Tek, Norcross, GA, USA) to extract total DNA in accordance with the
specific operation per the instructions of the kit. The Taqman probe was used for real-time
fluorescence quantitative PCR reaction, and RealMasterMix (Probe) was used for detection.
The number of copies contained in each sample was calculated by the Ct value and the
standard curve with each gram of content as the detection unit. The result was expressed
by the common logarithm of the number of bacteria in each gram of content (lg (copies/g)).
Animals 2023,13, 3006 5 of 14
Construction of the 20
µ
L reaction system: 8
µ
L 2
×
RealMasterMix, 1
µ
L of forward and
1
µ
L of reverse primers (100 nM), 1
µ
L 20
×
Probe Enhancer Solution, 0.3
µ
L probe, 1
µ
L
DNA, and 7.7
µ
L of RNase-Free ddH
2
O. Reaction procedure: pre denaturation at 95
◦
C for
10 s, denaturation at 95
◦
C for 15 s, annealing at 60
◦
C for 25 s, extension at 72
◦
C for 60 s,
40 cycles.
2.8. Gene Expression
About 0.1 g of the intestinal sample was put into a pre-cooled 1.5 mL centrifuge
tube, and 1 mL RNAiso Plus reagent (Takara, Kyoto, Japan) was added. The samples
were homogenized on ice until dissolved. After standing for 5 min, the samples were
centrifuged for 15 min at 12,000
×
g/4
◦
C. An amount of 400
µ
L supernatant was absorbed
into a new aseptic centrifuge tube, and isopropyl alcohol was added in the same volume,
inverted, and mixed gently. After standing on ice for 10 min, the samples were centrifuged
at
12,000×g/4 ◦C
for 10 min to precipitate RNA. The supernatant was discarded, and
1 mL of pre-cooled 75% ethanol (freshly mixed with DEPC water) was added, then the
suspended RNA was precipitated and centrifuged for 5 min at 7500
×
g/4
◦
C. Discard the
supernatant and leave it at room temperature for 1–2 min to dry the precipitate. When
the RNA was slightly dry, 40
µ
L DEPC water was added, and the pipette gun was used to
gently blow and suck 50 times to fully dissolve the RNA. By using a spectrophotometer
(NanoDrop 2000, Thermo Fisher Scientific, Inc., Waltham, MA, USA) to detect the purity
and concentration of total RNA, samples with an OD260/OD280 ratio of 1.8–2.0 were
considered suitable. Total RNA was used as template to synthesize cDNA by reverse
transcription kit, referring to the manual of PrimeScript
™
RT Reagent (Takara, Japan) for
specific operation. Reverse transcription consists of two steps: 42
◦
C, 2 min (removal of
genomic DNA); 37
◦
C for 15 min, 85
◦
C for 5 s. Based on the procedure and reaction
system of the TB Green
™
Premix Ex Taq
™
II (TaKaRa, Japan) kit, QuanStudio
™
6 Flex
real-time quantitative PCR was used to detect the expression of target genes in the small
intestine. The reaction system of qPCRas follows: TB Green Premix Ex Taq II (Tli RNaseH
Plus, 2
×
) 5
µ
L, ROX ReferenceDye II (50
×
) 0.2
µ
L, forward primer 0.4
µ
L, reverse primer
0.4 µL
, cDNA 1
µ
L, Double Steaming Water 3
µ
L. The reaction procedure as follows:
95 ◦C
predenaturation for 30 s, 95
◦
C denaturation for 5 s, 60
◦
C annealing for 34 s, 40 cycles.
Quantitative results used the GAPDH gene as the internal reference gene. The relative
expression levels of each target gene in tissues were calculated by2−∆∆Ct [34].
2.9. Data Statistics and Analysis
After all experimental data were analyzed by Microsoft Excel 2019, SPSS 24.0 statistical
software was used to perform one-way ANOVA on the experimental data of the three
treatment groups. When the differences were significant, Duncan’s multiple comparisons
were performed. All experimental data were expressed as “mean
±
standard error”,
p< 0.05 was considered significant, and 0.05 ≤p≤0.10 was considered a trend.
3. Result
3.1. Effect of Different Dietary Lipid Sources on Growth Performance and Nutrient Digestibility in
Weaned Pigs
Compared to the SO group, dietary supplementation of FPRO and CPRO had no
influence on ADFI and ADG (Table 2). The apparent digestibility of DM, EE, GE, and ash
was higher in the FPRO group than in the SO group (p< 0.05) (Table 3). Moreover, the
apparent digestibility of ash was also higher in the CPRO group than in the SO group
(p< 0.05).
Animals 2023,13, 3006 6 of 14
Table 2. Effects of different dietary lipids on growth performance of weaned piglets.
ITEM Treatments SEM p-Value
SO FPRO CPRO
Initial weight (kg) 7.91 7.52 7.97 0.11 0.231
Final weight (kg) 18.63 18.11 18.84 0.34 0.668
ADFI (g/d) 611.43 604.52 633.15 11.92 0.686
ADG (g/d) 362.96 378.27 380.19 9.47 0.736
F:G 1.69 1.60 1.66 0.02 0.436
ADFI, average daily feed intake; ADG, average daily gain; F:G, feed:gain ratio. (1) Mean and total SEM are listed
in separate columns (n = 8). (2) SO, pigs were fed with a basal diet; FPRO, pigs fed with 2% mixed oil 1; CPRO,
pigs fed with 2% mixed oil 2.
Table 3. Effects of different dietary lipids on nutrient digestibility of weaned piglets.
ITEM Treatments SEM p-Value
SO FPRO CPRO
DM (%) 80.35 b84.99 a83.41 ab 0.71 0.017
EE (%) 79.17 b84.22 a80.39 b0.62 0.001
GE (%) 80.93 b85.84 a83.15 ab 0.76 0.007
ASH (%) 55.65 b60.31 a59.42 a0.71 0.020
CP (%) 78.43 79.82 77.10 0.84 0.422
DM, dry matter; EE, ether extract; CP, crude protein; GE, Gross Energy. (1) Mean and total SEM are list in separate
columns (n = 8). (2) a, b mean values within a row with unlike superscript letters were significantly different
(p< 0.05
). (3) SO, pigs were fed with a basal diet; FPRO, pigs fed with 2% mixed oil 1; CPRO, pigs fed with 2%
mixed oil 2.
3.2. Effects of Different Dietary Lipid Sources on Serum Immunoglobulin, Pro-Inflammatory
Factors, and Antioxidant Capacity
As shown in Table 4, the serum IgA concentration in the FPRO group was higher than
that in the SO group (p< 0.05). In addition, the serum concentrations of pro-inflammatory
factors such as IL-6 and IL-1
β
in the FPRO and CPRO groups were lower than those in the
SO group (p< 0.05). Compared to the SO group, the serum CAT capacity was higher in the
FPRO and CPRO groups (p< 0.05).
Table 4.
Effects of different dietary lipids on serum indexes and antioxidant capacity of weanling piglets.
ITEM Treatments SEM p-Value
SO FPRO CPRO
IgA (µg/mL) 26.85 b31.18 a29.42 ab 0.70 0.032
IgG (µg/mL) 306.52 332.89 332.92 8.27 0.338
IgM (µg/mL) 13.65 14.71 14.43 0.24 0.203
IGF-1 (µg/L) 31.01 28.96 30.12 0.41 0.133
INS (mIU/L) 65.86 66.52 66.46 1.08 0.964
IL-6 (ng/L) 867.10 a762.34 b681.82 b24.24 0.016
IL-1β(ng/L) 33.12 a27.29 b27.00 b1.13 0.039
TNF-α(pg/mL) 277.83 243.12 273.70 8.28 0.155
Glucose (mmol/L) 5.47 4.86 5.82 0.18 0.108
Triglyceride (mol/L) 2.02 2.15 2.23 0.15 0.871
T-SOD (U/L) 62.69 65.58 65.99 1.14 0.472
AOC (U/mL) 2.89 3.23 2.79 0.33 0.861
MDA (nmol/mL) 3.88 4.20 4.45 0.21 0.547
GSH-PX (µmol/L) 674.96 1024.32 986.35 80.37 0.198
CAT (U/mL) 18.71 b39.80 a38.57 a3.49 0.014
(1) Mean and total SEM are list in separate columns (n = 8). (2) a, b mean values within a row with unlike
superscript letters were significantly different (p< 0.05). (3) SO, pigs were fed with a basal diet; FPRO, psigs fed
with 2% mixed oil 1; CPRO, pigs fed with 2% mixed oil 2.
Animals 2023,13, 3006 7 of 14
3.3. Effects of Different Dietary Lipid Sources on Intestinal Morphology and Mucosal
Enzyme Activities
As shown in Table 5and Figure 1, the FPRO and CPRO groups had a higher villus
height than the SO group in the jejunum and ileum, respectively (p< 0.05). The crypt
depth of the FPRO and CPRO group was also lower than that of the SO group (p< 0.05).
From Table 6, it can be seen that the maltase activity of the duodenal and jejunal mucosa
in the FPRO and CPRO groups is higher than in the SO group (p< 0.05). Compared
to the SO group, the lactase activity tended to be higher in the FPRO and CPRO group
(0.05 < p< 0.10).
Table 5. Effects of different dietary lipids on intestinal morphology of weaned piglets.
ITEM Treatments SEM p-Value
SO FPRO CPRO
Duodenum
Villus height (µm) 562.85 573.50 547.88 11.41 0.687
Crypt depth (µm) 297.50 295.27 293.96 7.66 0.985
V:C 1.92 1.86 1.88 0.04 0.639
Jejunum
Villus height (µm) 443.21 b463.16 b519.49 a11.36 0.009
Crypt depth (µm) 243.53 a201.61 b200.73 b7.10 0.021
V:C 2.18 2.13 2.02 0.038 0.418
Ileum
Villus height (µm) 547.32 b637.04 a542.90 b16.43 0.018
Crypt depth (µm) 317.15 284.84 277.49 9.12 0.272
V:C 1.92 2.02 1.86 0.03 0.178
V:C, Villus height:Crypt depth. (1) Mean and total SEM are list in separate columns (n = 8). (2) a, b mean values
within a row with unlike superscript letters were significantly different (p< 0.05). (3) SO, pigs were fed with a
basal diet; FPRO, pigs fed with 2% mixed oil 1; CPRO, pigs fed with 2% mixed oil 2.
Animals 2023, 13, x FOR PEER REVIEW 9 of 15
Figure 1. Effects of different dietary lipid sources on intestinal morphology (H&E; ×40).
3.4. Effects of Different Dietary Lipid Sources on Expression Levels of Key Genes Related to
Intestinal Epithelial Function and Mucosal Inflammatory Factors
As shown in Figure 2, the expression levels of tight junction protein ZO-1, Claudin-
1, and Occludin in the duodenal and ileal mucosa were significantly higher than that of
the SO and CPRO groups (p < 0.05). The expression levels of nutrient transporters such as
the CAT-1, PepT1, FATP1, and SGLT1 were higher in the FPRO group than in the SO group
(p < 0.05). At the same time, compared to the SO group, the expression level of IL-1β in
jejunum and ileum mucosa in FPRO and CPRO groups was significantly decreased (p <
0.05). The expression level of ERK1 in duodenum of FPRO group was also significantly
decreased p < 0.05), and the expression level of NF-κB in ileum mucosa of the CPRO group
was also significantly decreased (p < 0.05).
Figure 1. Effects of different dietary lipid sources on intestinal morphology (H&E; ×40).
Animals 2023,13, 3006 8 of 14
Table 6. Effects of different dietary lipids on intestinal mucosal enzymes in weaned piglets.
ITEM Treatments SEM p-Value
SO FPRO CPRO
Duodenum
AKP (U/g prot) 15.45 14.92 12.19 0.80 0.341
Maltase (ng/L) 303.96 b370.63 a335.14 ab 9.85 0.015
Lactase (pg/mL) 203.89 203.75 209.01 3.69 0.815
Sucrase (ng/L) 191.27 192.21 185.64 4.78 0.844
sIgA (µg/mL) 22.29 24.35 24.66 2.86 0.374
Jejunum
AKP (U/g prot) 21.33 b24.78 a23.60 ab 0.67 0.105
Maltase (ng/L) 120.63 b185.47 a187.18 a12.52 0.026
Lactase (pg/mL) 187.70 162.64 169.69 6.82 0.290
Sucrase (ng/L) 190.33 159.71 169.18 8.42 0.200
sIgA (µg/mL) 27.40 28.52 36.48 4.00 0.637
Ileum
AKP (U/g prot) 17.56 16.68 17.47 0.61 0.844
Maltase (ng/L) 353.42 355.95 359.95 7.20 0.938
Lactase (pg/mL) 283.35 318.31 322.21 7.86 0.079
Sucrase (ng/L) 243.25 240.86 255.44 4.71 0.418
sIgA (µg/mL) 5.62 4.33 4.83 0.58 0.684
APK, alkaline phosphatase. (1) Mean and total SEM are list in separate columns (n = 8). (2) a, b mean values
within a row with unlike superscript letters were significantly different (p< 0.05). (3) SO, pigs were fed with a
basal diet; FPRO, pigs fed with 2% mixed oil 1; CPRO, pigs fed with 2% mixed oil 2.
As shown in Table 7, the FPRO group had a higher abundance of Lactobacillus than
the SO group in the colon and cecum (p< 0.05). Compared to the SO group, the CPRO
group had a higher abundance of Lactobacillus in the cecum and a higher abundance of
Bifidobacterium in the colon, respectively (p< 0.05).
Table 7. Effects of different dietary lipids on intestinal microbial population in weaned piglets.
ITEM Treatments SEM p-Value
SO FPRO CPRO
Cecum, log (copies/g)
Escherichia coli 6.23 5.59 5.52 0.25 0.468
Lactobacillus 6.45 b7.29 a7.52 a0.17 0.018
Bifidobacterium 4.43 b4.74 ab 5.89 a0.29 0.089
Bacillus 8.24 8.25 8.17 0.06 0.868
Colon, log (copies/g)
Escherichia coli 5.45 5.40 5.18 0.20 0.859
Lactobacillus 6.82 b7.91 a7.33 ab 0.19 0.070
Bifidobacterium 4.49 b4.63 b6.18 a0.25 0.025
Bacillus 8.49 8.39 8.31 0.05 0.440
(1) Mean and total SEM are list in separate columns (n = 8). (2) a, b mean values within a row with unlike
superscript letters were significantly different (p< 0.05). (3) SO, pigs were fed with a basal diet; FPRO, pigs fed
with 2% mixed oil 1; CPRO, pigs fed with 2% mixed oil 2.
3.4. Effects of Different Dietary Lipid Sources on Expression Levels of Key Genes Related to
Intestinal Epithelial Function and Mucosal Inflammatory Factors
As shown in Figure 2, the expression levels of tight junction protein ZO-1, Claudin-1,
and Occludin in the duodenal and ileal mucosa were significantly higher than that of the
SO and CPRO groups (p< 0.05). The expression levels of nutrient transporters such as the
CAT-1, PepT1, FATP1, and SGLT1 were higher in the FPRO group than in the SO group
(p< 0.05)
. At the same time, compared to the SO group, the expression level of IL-1
β
in jejunum and ileum mucosa in FPRO and CPRO groups was significantly decreased
Animals 2023,13, 3006 9 of 14
(p< 0.05)
. The expression level of ERK1 in duodenum of FPRO group was also significantly
decreased p< 0.05), and the expression level of NF-
κ
B in ileum mucosa of the CPRO group
was also significantly decreased (p< 0.05).
Animals 2023, 13, x FOR PEER REVIEW 10 of 15
Figure 2. The expression levels of tight junction proteins. a, b mean values within a row with
unlike superscript leers were significantly different (p < 0.05)
4. Discussion
Fat and oil are commonly considered as effective energy sources for animals,
including pigs [35]. The digestion and metabolism of lipids are dependent on their
sources, dietary proportions, and intermolecular distribution of saturated and
unsaturated fay acids [36,37]. Previous studies on the weaned pigs (5 to 20 kg) indicated
that dietary fat supplementation reduced feed intake, but significantly improved the feed
conversion efficiency [8]. Compared to other vegetable oils, palm oil and coconut oil are
mainly composed of MCFA (the fay acids below 16 carbon atoms in coconut oil account
for 66.2% of the total fay acids) [38], and their digestibility levels range from 80 to 95%
Figure 2.
The expression levels of tight junction proteins. a, b mean values within a row with unlike
superscript letters were significantly different (p< 0.05).
4. Discussion
Fat and oil are commonly considered as effective energy sources for animals, includ-
ing pigs [
35
]. The digestion and metabolism of lipids are dependent on their sources,
dietary proportions, and intermolecular distribution of saturated and unsaturated fatty
acids [
36
,
37
]. Previous studies on the weaned pigs (5 to 20 kg) indicated that dietary fat
Animals 2023,13, 3006 10 of 14
supplementation reduced feed intake, but significantly improved the feed conversion effi-
ciency [
8
]. Compared to other vegetable oils, palm oil and coconut oil are mainly composed
of MCFA (the fatty acids below 16 carbon atoms in coconut oil account for 66.2% of the total
fatty acids) [
38
], and their digestibility levels range from 80 to 95% [
39
]. Long-chain fatty
acids can delay stomach emptying and increase feelings of fullness, which may decrease
the feed intake for pigs [
40
]. In the present study, we discussed the effects of different lipid
mixtures on the growth performance and intestinal health of weaned piglets. As expected,
the digestibility levels of DM, GE, and ash were higher in the FPRO and CPRO group than
in the SO. Moreover, the FPRO group had a higher digestibility of EE than other groups,
which may be associated with the presence of a considerable amount of unsaturated fatty
acids in the fish oil [41].
Immunoglobulins, also known as antibodies, are glycoprotein molecules produced by
plasma cells, which act as a critical part of the immune response by specifically recognizing
and binding to particular antigens, such as bacteria or viruses, and aiding in their destruc-
tion [
42
]. In this study, dietary supplementation of FPRO can significantly increase the
serum IgA concentration, indicating an elevated immunity in the pigs. Pro-inflammatory
cytokines, such as IL-1
β
and IL-6, can mediate host inflammatory processes and produce a
rapid immune response after infection with pathogenic microorganisms [
43
]. In this study,
the serum IL-1
β
and IL-6 concentrations were decreased in the FPRO and CPRO group.
This is probably due to the fish and coconut oil in the two lipid mixtures, as previous
studies indicated that dietary supplementation of coconut oil and fish oil can reduce the
occurrence of inflammatory responses in the weaned pigs [
44
,
45
]. Interestingly, the serum
activity of CAT was also higher in the FPRO and CPRO group than in the SO group. CAT,
as a key enzyme in the antioxidant defense system, plays an important role in clearing hy-
drogen peroxide in the body and avoiding oxidative stress [
46
]. A previous study indicated
that palm oil contains a large number of antioxidants, which can improve the activity of
antioxidant enzymes [
47
], whereas rice oil contains a large number of compounds with
antioxidant properties, such as glutamine, tocopherol, and tocotrienol [21].
The digestion and absorption of various nutrients are basically carried out in the small
intestine. Villus height and crypt depth of intestinal mucosal epithelium are closely related
to the absorption capacity of intestinal mucosa [
48
]. In this study, the villus height of
the jejunum and ileum in the FPRO and CPRO groups was higher than in the SO group,
respectively, and the crypt depth in FPRO and CPRO groups was also lower than in the
SO group. Coconut oil and palm oil are rich in medium-chain fatty acids [
49
], MCFA can
be directly utilized by intestinal epithelial cells for energy production, thereby helping
to support the integrity of the piglets’ intestines [
50
]. Previous studies have shown that
MCFA supplementation for the weaned pigs can improve the intestinal morphology and
integrity, as indicated by the significant increase in intestinal villus length, and decrease
in crypt depth and the number of intraepithelial lymphocytes [
51
]. Fish oil has also been
reported to increase the villus height and villus height/crypt depth ratio in the weaned
pigs [
6
]. Changes of intestinal morphology are usually accompanied by brush margins of
intestinal epithelium digestive enzyme activity [
52
]. In this study, the maltase and lactase
activities of duodenal and jejunal mucosa in FPRO and CPRO groups were higher than
that in the SO group.
The gut microbiota is an important component of the gut ecosystem, and is closely
related to the integrity of intestinal epithelial cells, the digestion and metabolism of nu-
trients, immune responses, and diseases [
17
,
53
]. Some difficult-to-digest fats can reach
the hindgut and be a source of nutrients for bacteria, which may regulate the gut bacteria
balance [
54
]. Different sources of oils have different compositions, so their effects on gut
microbes are also different [
55
]. In this study, the abundance of lactobacillus in the colon
and cecum in the FPRO group was higher than in the SO group. Moreover, the abundance
of cecal lactobacillus i and colonic bifidobacterium in the CPRO group was also higher
than in the SO group. This result is consistent with previous studies that found that dietary
Animals 2023,13, 3006 11 of 14
supplementation of rice and coconut oil significantly increased the abundances of beneficial
bacteria such as bifidobacterium and Lactobacillus in the intestine [56,57].
Tight junctions play a crucial role in maintaining intestinal permeability, consisting
of transmembrane barrier proteins (such as claudin and occludin), cytoplasmic scaffold
proteins (such as the ZO family), and adhesion molecules [
58
]. A previous study indicated
that fish oil can enhance the expression levels of occludin and claudin-1 in the weaned
pigs [
6
]. In this study, the expression levels of ZO-1, Claudin-1, and Occludin in the
duodenum and ileum were higher in the FPRO group than in the SO and CPRO group.
Moreover, the expression levels of nutrient transporters such as the CAT-1, PepT1, FATP1,
and SGLT1 were higher in the FPRO group than in the SO group. PepT1 is a small peptide
transporter, while CAT1 is mainly responsible for the transport of basic amino acids [
59
,
60
].
GLUT-2 is one of the major transporters of glucose absorption, while FATP-1 is responsible
for the transport of long-chain fatty acids into intestinal cells [
61
]. These results indicated
improved epithelium functions in the weaned pigs upon FPRO supplementation. IL-1
β
is
considered to be an important pro-inflammatory cytokine involved in innate immunity;
in general, down-regulating the expression of IL-1
β
protein in intestinal tissue can reduce
inflammatory damage [
62
]. NF-
κ
B is a promoter of inflammation-associated tumors, and it
is essential for promoting inflammation-associated cancers [
63
]. Previous studies indicated
that dietary supplementation of coconut oil and fish oil can reduce the occurrence of
inflammatory responses in the weaned pigs [
44
,
45
]. In this study, the expression levels
of IL-1
β
in the jejunum and ileum were down-regulated in the FPRO and CPRO group.
Moreover, the expression levels of duodenal ERK1 and ileal NF-
κ
B were decreased in the
FPRO and CPRO groups, respectively. These results may suggest an anti-inflammatory
effect of a lipid mixture containing the fish and coconut oil.
5. Conclusions
In the present study, we explored the effects of different lipid sources on growth
performance and intestinal health in weaned pigs. We observed improved digestibility and
intestinal epithelium functions, as well as a reduced production of inflammatory cytokines
in the FPRO and CPRO groups, which may offer a theoretical basis for rational selection of
lipid sources for the weaned pigs as well as other mammalian animals. Of course, this study
has a relatively short duration and may have certain limitations. Further experimental
verification is needed regarding the effects of mixed oil FPRO and CPRO on fattening pigs
and other animals.
Author Contributions:
J.H. conceived and designed the experiments. W.Y. and F.J. conducted
the experiment. B.Y., Y.L., A.W., P.Z., Z.H., J.Y. and X.M. analyzed the data. H.Y. and J.L. made
the final editing and proofreading. All authors have read and agreed to the published version of
the manuscript.
Funding:
This work was funded by the National Natural Science Foundation of China (no. 31972599)
and the Development Program of Sichuan Province (2020YFN0147).
Institutional Review Board Statement:
All experimental protocols used in the animal experiment
were approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural Univer-
sity (No. 20181105).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data used to support the findings of this study are available from
the corresponding author upon request.
Acknowledgments:
We would like to thank Fujian Longyan Singao Company for providing the test
site and Limei Sun for her help in the animal testing and sample collection process.
Conflicts of Interest: The authors declare no conflict of interest.
Animals 2023,13, 3006 12 of 14
Abbreviations
ADFI average daily feed intake
ADG average daily body weight gain
CAT Catalase
CP crude protein
DAO diamine oxidase
DM dry matter
EE ether extract
G/F the gain-to-feed ratio
GE gross energy
GLUT2 glucosetransporter-2
GSH-Px Glutathione peroxidase
IgA immunoglobulins A
IgG immunoglobulins G
IgM immunoglobulins M
MDA Malondialdehyde
V:C Villus height:Crypt depth
FATP Fatty acid transport proteins
IL-1βInterleukin1-β
IL-6 Interleukin-6
SGLT-1 Sodium/glucose cotransporter-1
ZO-1 zonula occludens
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