RESEARCH ARTICLE

Host-Microbe Biology

Consumption of Dietary Fiber from Different Sources during

Pregnancy Alters Sow Gut Microbiota and Improves
Performance and Reduces Inflammation in Sows and Piglets
Boshuai Liu,a Xiaoyan Zhu,a,b Yalei Cui,a,b Wenjing Wang,a Hua Liu,a Zidan Li,a Zhiguo Guo,a Sen Ma,a,b Defeng Li,a,b
Chengzhang Wang,a,b Yinghua Shia,b

College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, China
a

b Henan Key Laboratory of Innovation and Utilization of Grassland Resources, Zhengzhou, China

Boshuai Liu and Xiaoyan Zhu contributed equally to this article. Author order was determined alphabetically.

ABSTRACT In pregnant and lactating sows, metabolism and immunity undergo

drastic changes, which can lead to constipation, abortion, and intrauterine growth
restriction (IUGR) and reduce production performance. Dietary fiber can regulate ani-
mal gut microbiota, alleviate inflammatory responses, and improve performance.
Here, 48 sows (Large  Landrace) were randomly allocated to groups including, con-
trol, and with alfalfa meal (AM), beet pulp, and soybean skin dietary supplementa-
tion for 60 days of gestation. The AM diet decreased IUGR, increased food intake dur-
ing lactation, and promoted the reproductive performance and physical condition of
sows. Further, the AM diet significantly reduced markers of intestinal permeability
(reactive oxygen species and endotoxin) in sow serum, and of systemic inflammation
(interleukin-6 [IL-6] and tumor necrosis factor alpha) in sow feces and serum, as well
as piglet serum, while it increased the anti-inflammatory marker, IL-10, in sow serum

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and feces. The AM diet also significantly affected gut microbiota by increasing the
relative abundance of proinflammatory bacteria, while decreasing anti-inflammatory
bacteria. Moreover, the total short-chain fatty acid (SCFA) content was higher in
feces from sows fed an AM diet, with butyric acid content significantly higher during
lactation, than in controls. Sow performance was correlated with intestinal perme-
ability, inflammation, and gut microbiota, which were also vertically transmitted to
piglets. Our results are significant for guiding feed management in the pig breeding
industry. Further, the “sows to piglets” model provides a reference for the effect of
dietary fiber on the gastrointestinal function of human mothers and infants.
Citation Liu B, Zhu X, Cui Y, Wang W, Liu H, Li
IMPORTANCE Although the direct effects of dietary fiber on gut microbiota compo- Z, Guo Z, Ma S, Li D, Wang C, Shi Y. 2021.
sition have been studied extensively, systematic evaluation of different fiber sour- Consumption of dietary fiber from different
sources during pregnancy alters sow gut
ces on gut health and inflammatory responses of sows and their offspring has microbiota and improves performance and
rarely been conducted. Excessive reactive oxygen species produced by overactive reduces inflammation in sows and piglets.
metabolic processes during late pregnancy and lactation of sows leads to increased mSystems 6:e00591-20. https://doi.org/10
.1128/mSystems.00591-20.
endotoxin levels, disordered gut microbiota, decreased SCFA production, and
Editor Mariana X. Byndloss, Vanderbilt
secretion of proinflammatory factors, which in turn causes local inflammation of University Medical Center
the gut, potential damage of the gut microbial barrier, increased gut permeability, Copyright © 2021 Liu et al. This is an open-
increased blood endotoxin levels (resulting in systemic inflammation), and ulti- access article distributed under the terms of
the Creative Commons Attribution 4.0
mately decreased sow and piglet performance. Our results showed that supple- International license.
mentation of the diet with alfalfa meal in mid and late pregnancy can reverse this Address correspondence to Yinghua Shi,
process. Our findings lay a foundation for improving the gut health of sows and [email protected].
piglets and provide insights into the study of the gastrointestinal tract function in Received 30 June 2020
human mothers and infants. Accepted 5 January 2021
Published 26 January 2021
KEYWORDS dietary fiber, inflammation, gut microbiota, sows, piglets, animal nutrition

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Liu et al.

S ows and piglets are excellent animal models and have been widely used in bio-
medical research. Compared to rodents, sows and piglets are considered a superior
model for studying the relationships in gut function of human mothers and infants (1).
Pigs have many characteristics similar to humans, including digestive physiology,
microbiota, and diet. These animals are suitable for a multitude of disease models,
including diarrhea, gastrointestinal inflammatory disorders, necrotizing enterocolitis of
neonates, and obesity, etc. (2). In large-scale pig production, sows and piglets are cru-
cial to determining production levels and the economic benefits of pig farms, and the
gestational, lactation, and newborn periods are core stages for feed management of
sows and piglets in large-scale pig production (3). During pregnancy, sows undergo
dramatic changes in physiological metabolism and immunity to ensure the implanta-
tion and development of embryos and pregnancy completion (4). In the mid and late
periods of pregnancy, the levels of tumor necrosis factor alpha (TNF-a), interleukin-6
(IL-6), reactive oxygen species (ROS), and other proinflammatory factors increase signif-
icantly in the blood of sows (5) and are closely related to numerous diseases, including
constipation, abortion, and intrauterine growth retardation (IUGR) (6, 7). Further,
expression of the tight-junction protein, zonulin, is increased in sow guts, while bacte-
rial lipopolysaccharide (LPS) entering the circulation through the gut barrier increased,
and increased concentrations of bacterial endotoxins in the circulation can lead to
metabolic endotoxemia, which is a potential mediator of inflammation (8–10). To initi-
ate and maintain lactation, sows undergo complex metabolism and immune system
changes that directly affect the development and growth of piglets (11). Therefore,
reduction of inflammatory responses and ensuring normal metabolic and immune
changes in sows during mid and late pregnancy and lactation is crucial for the perform-
ance of sows and their offspring (4). Gut microbiota has key roles in nutrient metabolism,
immune development, protection against pathogens, and the pathogenesis of many
chronic diseases in the host (12–14). Sow gut microbiota change dramatically during
pregnancy and may be involved in metabolic processes in pregnant animals (15).
Compared to the early stage of pregnancy, Proteobacteria and Actinobacteria are signifi-
cantly increased in the sow gut during the late stage of pregnancy and have clear char-

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acteristics associated with increased risk of inflammation and energy loss (15). Short-
chain fatty acids (SCFAs) are the main fermentation metabolites of gut microbiota and
can stimulate cell signal transduction pathways via G protein-coupled receptor and up-
regulate the expression of Toll-like receptors (16). SCFAs also inhibit the activity of his-
tone deacetylases (HDACs) and mRNA expression levels of the nuclear transcription fac-
tor, NF-κB, as well as downregulate the production of proinflammatory factors, to reduce
gut inflammation (17). In addition, sow gut microbiota also participates in the immune
development and maturation of their offspring. Transplanting microbiota colonized for a
short period of time during the gestation period of a sow to germfree mice can promote
the development of innate immunity in the gut and reduce inflammatory responses in
their offspring via the activity of microbiota and metabolites (18). The SCFAs produced
by sow gut microbiota can also be transferred to their offspring, where they promote
the maturation and development of the immune system (19). Therefore, it is vital to
understand the role of gut microbiota and their SCFA metabolites in the changes in
inflammatory responses in sows during mid and late pregnancy and lactation.
Interest regarding the beneficial role of dietary fiber in regulating gut microbiota and
physiological inflammatory responses is currently growing rapidly (20). Meanwhile, the
advantages of dietary fiber in sows have gradually been exploited, e.g., by improving
oocyte quality, increasing early embryo survival, improving lactation and weaning per-
formance, and enhancing the vitality and uniformity of newborn piglets (21). This is pri-
marily because dietary fiber can increase beneficial microbes in the gut, particularly lac-
tobacilli, which aid in digestion and gut barrier function (22). In addition, SCFAs are the
main fermentation products of dietary fiber and play an important role in gut health (23,
24). For example, acetate is an anti-inflammatory metabolite that maintains gut homeo-
stasis, while butyrate helps to regulate gut permeability (25).

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Gut Microbiota and Inflammation in Sows and Piglets

TABLE 1 Effects of different fiber sources on fat thickness and feed intake of sows
Mean ± SDa

Description CK AM BP SH
Day 60 in gestation (mm) 19.82 6 1.00 19.83 6 1.35 19.83 6 1.16 19.68 6 0.84
Day 90 in gestation (mm) 19.54 6 0.78 19.77 6 1.22 19.83 6 1.08 19.73 6 0.61
Before parturition (mm) 20.84 6 2.37 20.43 6 1.43 20.72 6 1.35 19.77 6 0.80
After weaning (mm) 19.38 6 1.54 20.53 6 0.94 19.67 6 2.11 19.15 6 2.23
Gain during gestation (mm) 1.12 6 1.76 0.65 6 0.74 0.95 6 2.83 0.22 6 1.01
Loss during lactation (mm) 1.43 6 3.78 –0.09 6 0.16 1.20 6 1.87 0.62 6 2.44
Pregnancy feed intake (kg) 2.44 6 0.27 2.37 6 0.26 2.35 6 0.39 2.46 6 0.38
Lactation feed intake (kg) 7.75 6 0.66B 8.76 6 0.44A 7.80 6 0.64B 7.66 6 0.59B
aData for the control (CK), alfalfa meal (AM), beet pulp (BP), and soybean skin (SH) groups are presented. The
data were evaluated by one-way ANOVA, and significant differences between means were assessed by using
Duncan’s test. Differences in the superscript letters for peer data indicate that a difference is significant (P ,
0.05). The lack of a superscript letter means that all differences were nonsignificant (P . 0.05).

The effects of dietary fiber vary greatly due to their different sources and the variety
and complexity of their chemical structures (26). Darroch et al. (27) added 20% soy-
bean hulls and 0.3% psyllium to the diets of pregnant sows. Their results showed that
soybean hull was more conducive to physical health maintenance in pregnant sows
but had little effect on litter size. Cheng et al. (20) added combined soluble fiber from
pregelatinized waxy corn starch and guar gum to the sows’ pregnancy diet, which sig-
nificantly improved the developmental growth performance and gut function of 14-
day-old suckling piglets. Zhuo et al. (28) found that insoluble fiber oat bran mixed with
corn or soybean meal produced more SCFA by gut fermentation, which improved pig
behaviors and reproductive performance. Although the direct effects of dietary fiber
on gut microbiota composition have been studied extensively, systematic evaluation
of different fiber sources (e.g., insoluble and soluble fiber sources) on gut health and
inflammatory responses of sows and their offspring during mid and late gestation pe-
riod has rarely been conducted. In this study, we evaluated the effects of adding differ-
ent fiber sources, including soybean husk (SH), alfalfa meal (AM), and beet pulp (BP), to

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sow diets on growth performance, gut microbiota, gut permeability, and inflammation
in sows and piglets. Our findings lay the foundation for screening specific fiber sources
to improve the performance and gut health of sows and piglets and provide insights
into the study of the gastrointestinal tract function in human mothers and infants.

RESULTS
Effects of supplementation in mid to late pregnancy with dietary fiber from
different sources on the performance of sows and piglets. Sows (n = 48; Large 
Landrace) at 60 days of gestation were randomly allocated to groups as follows: control
animals (CK) and animals with dietary supplementation using alfalfa meal (AM), beet
pulp (BP), and soybean skin (SH). Assessment of sow backfat thickness during the
reproductive cycle, including at gestation day 60 (G60d), G90d, lactation day 0 (L0d),
and L21d, and sow feed intake by one-way analysis of variance (ANOVA) (Table 1)
showed that there was no significant difference in average daily feed intake (P =
0.7620) and backfat (P = 0.6290 and P = 0.4240, respectively) among the treatment
groups in mid to late pregnancy; however, in the lactation period, the average daily
feed intake (P = 0.0320) of the AM group was significantly higher than that of CK, BP,
and SH groups; in addition, the backfat loss of lactating sows showed a downward
trend (P = 0.3750). Evaluation of sow reproductive performance demonstrated no sig-
nificant differences in total litter size (P = 0.1380), live litter size (P = 0.1510), newborn
body weight (P = 0.4430), or newborn litter weight (P = 0.1170) among the treatment
groups; however, IUGR was significantly lower in the AM group than in the CK, BP, and
SH groups (P = 0.0370) (Table 2). Further, we conducted a systematic study of piglet
growth performance during lactation (L7d, L14d, and L21d) and found that individual
body weight (P = 0.0700, P = 0.1580, and P = 0.0980, respectively) and litter weight

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TABLE 2 Effects of different fiber sources on reproductive performance of sows

Mean ± SDa

Description CK AM BP SH
Total born piglets 13.11 6 2.85 12.13 6 3.31 10.44 6 4.28 13.11 6 2.62
Live-born piglets 12.67 6 2.83 12.00 6 3.42 10.11 6 4.28 12.56 6 2.46
IUGR (%) 9.48 6 0.07A 2.08 6 0.04B 11.21 6 0.08A 8.94 6 0.06A
Body wt of newborn piglets (kg) 1.35 6 0.17 1.37 6 0.12 1.40 6 0.17 1.42 6 0.17
Litter wt at birth (kg) 16.81 6 2.42 16.49 6 4.86 14.04 6 5.73 17.72 6 3.79
aData for the control (CK), alfalfa meal (AM), beet pulp (BP), and soybean skin (SH) groups are presented. The
data were evaluated by one-way ANOVA, and significant differences between means were assessed by using
Duncan’s test. Differences in superscript letters for peer data indicate that a difference is significant (P , 0.05).
The lack of a superscript letter means that all differences were nonsignificant (P . 0.05).

(P = 0.0014, P = 0.1580, and P = 0.0980, respectively) of piglets in the AM, BP, and SH
groups showed an increasing trend compared to the CK (Table 3). These data indicate
that AM intake during mid to late pregnancy can increase feed intake and reduce IUGR
in lactating sows.
Effects of AM diet on gut permeability and inflammatory responses in sows
and piglets. Given our findings that AM supplementation could reduce IUGR, we next
conducted a systematic evaluation of three biomarkers related to gut permeability
(ROS, endotoxin, and zonulin) by one-way ANOVA at G100d, L4d, and L18d in sows
with dietary supplementation with AM compared to controls. Compared to CK sows,
the ROS (P = 0.0004, P , 0.0001, and P = 0.0010, respectively) and endotoxin (P =
0.0100, P = 0.0018, and P = 0.0007, respectively) levels were significantly lower at
G100d, L4d, and L18d in serum samples from sows fed an AM diet (Fig. 1A and C).
Further, serum levels of zonulin at L18d were significantly lower than those in the CK
(P = 0.0014) (Fig. 1B). Together, these data suggest that AM intake in mid to late preg-
nancy reduces sow gut permeability. Next, we examined four biomarkers associated
with gut inflammation in sows, IL-6, lipocalin-2, TNF-a, and IL-10. In terms of systemic
inflammatory responses, we found that serum levels of IL-6 (P = 0.0014, P = 0.0010,
and P , 0.0001, respectively), lipocalin-2 (P = 0.0122, P = 0.0039, and P = 0.0044,

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respectively), and TNF-a (P = 0.0012, P = 0.0008, and P = 0.0084, respectively) were sig-
nificantly reduced, while those of IL-10 (P = 0.0040, P = 0.0025, and P = 0.0014, respec-
tively) were significantly increased in sows fed a AM diet at G100d, L4d, and L18d, com-
pared to controls in the CK (Fig. 1D to G). In sow feces, endotoxin levels were
significantly lower at L4d and L18d (Fig. 1H). Evaluation of gut inflammatory responses

TABLE 3 Effects of different fiber sources on piglet performance

Mean ± SDa (n = 11)

Description CK AM BP SH
Litter wt (kg)
Day 0 19.48 6 2.24 19.42 6 2.02 19.44 6 2.89 19.53 6 1.23
Day 7 39.82 6 1.74 39.87 6 4.33 43.53 6 4.61 42.76 6 4.16
Day 14 54.90 6 3.77 54.91 6 2.38 57.59 6 5.25 57.51 6 6.39
Day 21 76.16 6 5.80 82.56 6 1.15 79.64 6 6.66 75.93 6 7.60
Avg daily gain 2.83 6 0.29 3.16 6 0.09 3.01 6 0.20 2.82 6 0.34

Body wt (kg)
Day 0 1.38 6 0.20 1.38 6 0.18 1.38 6 0.26 1.39 6 0.11
Day 7 3.62 6 0.16 3.62 6 0.39 3.96 6 0.42 3.89 6 0.38
Day 14 4.99 6 0.34 4.99 6 0.22 5.24 6 0.48 5.00 6 0.66
Day 21 6.92 6 0.53 7.51 6 0.10 7.24 6 0.61 6.90 6 0.69
Avg daily gain 0.26 6 0.03 0.29 6 0.01 0.27 6 0.01 0.26 6 0.03
aData for the control (CK), alfalfa meal (AM), beet pulp (BP), and soybean skin (SH) groups are presented. Eleven
animals were included in each group. The data were evaluated by one-way ANOVA, and significant differences
between means were assessed by using Duncan’s test. Differences in superscript letters for the peer data
indicate that a difference is significant (P , 0.05). The lack of a superscript letter means that all differences were
nonsignificant (P . 0.05).

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FIG 1 Effect of alfalfa meal diet on serum ROS (A), serum zonulin (B), serum endotoxin (C), serum
lipocalin-2 (D), serum IL-6 (E), serum TNF-a (F), serum IL-10 (G), fecal endotoxin (H), fecal IL-6 (I), fecal
TNF-a (J), and fecal IL-10 (K) levels of sows. CK, control group; AM, alfalfa meal group. The data were
evaluated by one-way ANOVA, and significant differences between means were assessed by using
Duncan’s test. *, 0.01 , P # 0.05; **, 0.001 , P # 0.01; ***, P # 0.001; ns . 0.05.

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Liu et al.

FIG 2 Effect of alfalfa meal diet on serum ROS (A), serum endotoxin (B), serum IL-6 (C), serum TNF-a (D) and
serum IL-10 (E) levels of piglets. CK, control group; AM, alfalfa meal group. The data were evaluated by one-
way ANOVA, and significant differences between means were assessed by using Duncan’s test. *, 0.01 , P #
0.05; **, 0.001 , P # 0.01; ***, P # 0.001; ns, P . 0.05.

demonstrated that levels of IL-6 (P = 0.0010 and P = 0.0017, respectively) and TNF-a
(P = 0.0052 and P = 0.0084, respectively) were significantly reduced at L4d and L18d in
fecal samples from sows fed with an AM, while those of IL-10 (P = 0.0130 and P =

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0.0443, respectively) were significantly increased (Fig. 1I, J, and K).
Further, we assessed these biomarkers by one-way ANOVA in the sera of piglets to
understand whether feeding sows with an AM supplemented diet is related to gut per-
meability and systemic inflammatory responses in suckling piglets. We found no signif-
icant differences at L4d. At L18d, the serum levels of ROS (P = 0.0207), endotoxin (P =
0.0211), IL-6 (P = 0.0001), and TNF-a (P = 0.0067) were significantly reduced in AM
group, while the serum levels of IL-10 (P = 0.0094) were significantly increased (Fig. 2).
These results indicated that sows with alfalfa meal supplementation had lower levels
of gut permeability biomarkers and lower levels of inflammatory markers in sows and
piglets.
An AM diet regulates changes in sow gut microbiota composition and microbial
metabolites. The microbiota in sow fecal samples were analyzed at three time
points (G100d, L4d, and L18d) by deep sequencing of the bacterial 16S rRNA gene
V3-V4 region (see Fig. S1A in the supplemental material). The AM diet had no effect
of on the Shannon (P = 0.1626, P = 0.5106, and P = 0.2304, respectively) and Chao
(P = 0.2505, P = 0.8903, and P = 0.5451, respectively) indices by a Wilcoxon rank
sum test in sow fecal microbiota at any time point (G100d, L4d, or L18d) (see
Fig. S1B and C). Community composition at the phylum level indicated that indi-
cated that the dominant microbiota at the three stages were Firmicutes (61.7 to
73.7%), Bacteroidetes (18.3 to 27.7%), Spirochaetae (3.6 to 7.6%), and Proteobacteria
(0.5 to 4.5%) (see Fig. S1D), while dominant genera were Clostridium_sensu_stricto_1 (5.7
to 13.2%), norank_f_Bacteroidales_S24-7_group (5.5 to 9.1%), Terrisporobacter (3.8 to 9.9%),
Christensenellaceae_R-7_group (3.2 to 8.7%), and Lactobacillus (1.5 to 13.7%), among others
(see Fig. S1E). Principal-component analysis by Bray-Curtis and unweighted-UniFrac
distance showed that there were significant differences in microbiota at G100d, L4d, and
L18d between the CK and AM groups (Fig. 3A). Further linear discriminant analysis effect

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FIG 3 AM diet regulates the changes of gut microbiota composition in sows. (A) Principal-component analysis of OTU level by
Bray-Curtis and unweighted-UniFrac distance; (B) LEfSe analysis determined by one-against-all (less strict). CK, control group;
AM, alfalfa meal group.

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FIG 4 Effect of alfalfa meal diet on SCFAs fermentation of sows. (A) Fecal amino acids (AA); (B) fecal propionic
acid (PA); (C) fecal butyric acid (BA); (D) fecal total SCFAs. The data were evaluated by one-way ANOVA, and
the significant differences between means were assessed by using Duncan’s test. *, 0.01 , P # 0.05; **,
0.001 , P # 0.01; ***, P # 0.001; ns, . 0.05.

size (LEfSe) analysis by “one-against-all” (less strict) to evaluate differences between the
two groups showed that, at G100d, there was a significant increase in the relative abun-

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dance of Prevotellaceae_NK3B31_group, Lachnoclostridium_1, Eubacterium_eligens_group,
Paraprevotella, norank_fs_p_2534_18b5_gut_group, and Clostridium_sensu_stricto_6 and
reduction in the relative abundance of Helicobacter, Terrisporobacter in animals fed
the AM supplemented diet compared to the CK. At L4d, there was a significant
increase in the relative abundance of Lachnospiraceae_NK4A136_group and a reduced
relative abundance of Desulfovibrio in the AM diet group compared to the CK. Finally, at
L18d, the AM diet led to a significant increase in the relative abundance of Clostridium_
sensu_stricto_1 and a reduction in unclassified_f_Lachnospiraceae, Eubacterium_
fissicatena_group, Erysipelotrichaceae_UCG_004, and Ruminococcaceae_V9D2013_group
relative to the CK (Fig. 3B). These results indicate that feed supplemented with AM during
pregnancy significantly changes the gut microbiota composition in sows.
To analyze the effect of an AM diet on gut microbial metabolism in sows during
pregnancy, we next studied SCFAs by one-way ANOVA in sow excrement at three time
points: G100d, L4d, and L18d (Fig. 4). The results showed that compared to the CK, all
SFCAs in the feces of sows fed an AM supplemented diet had an increasing trend.
There was no statistically significant except butyrate of L4d (P = 0.0360).
Sow performance is related to gut permeability and inflammatory responses,
which influence the health and growth of piglets. Spearman correlation analysis by
Euclidean distance found that sow performance was correlated with gut permeability
and inflammation (Table 4), and IUGR was positively correlated with serum lipocalin-2,
IL-6, and TNF-a and significantly negatively correlated with serum IL-10.
Spearman correlation analysis by Euclidean distance found that sow feed intake
during lactation was negatively correlated with serum lipocalin-2, IL-6, and TNF-a, as
well as fecal IL-6 and serum ROS, and significantly positively correlated with serum IL-
10. Gut permeability was correlated with inflammation in both sows and piglets
(Table 5). In piglets, serum IL-10 was positively correlated with sow serum IL-10 and

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TABLE 4 Correlation between IUGR, lactation feed intake, and inflammatory factors of sows
Correlationa

Description Serum lipocalin-2 Serum IL-6 Serum TNF-a Serum IL-10 Fecal IL-6 Fecal TNF-a Fecal IL-10 Serum ROS
IUGR (L4d) 0.864* 0.914* 0.926** –0.923** 0.773 0.363 –0.616 0.840
Lactation feed intake (L18d) –0.649* –0.638* –0.695* 0.685* –0.745** –0.398 0.201 –0.806**
aThedata were evaluated by Spearman correlation analysis of the Euclidean distance. *, Mean significant correlation (P , 0.05); **, mean extremely significant correlation
(P , 0.01).

significantly negatively correlated with sow fecal TNF-a. Further, piglet serum TNF-a
levels were positively correlated with serum IL-6, TNF-a, and ROS, as well as fecal IL-6
in sows, and significantly negatively correlated with piglet weaning body weight.
Serum IL-6 in piglets was positively correlated with serum lipocalin-2, IL-6, TNF-a, and
ROS in sows, while there was no significant correlation between serum ROS in piglets
and sow inflammatory factors or piglet weaning body weight.
The composition of gut microbiota of sows regulated by an AM supplemented
diet is related to their gut health. As shown in Fig. 5A, analysis of the correlation
between microbiota differing according to LEfSe and metabolic indices in the gut tract
of sows at each stage showed that the Prevotellaceae_NK3B31_group was significantly
positively correlated with serum IL-10, while the norank_f_2534-18b5_gut_group was
significantly positively correlated with serum IL-10 and fecal IL-10 and negatively corre-
lated with serum lipocalin-2, IL-6, and TNF-a. Terrisporobacter was positively correlated
with serum lipocalin-2, TNF-a, and endotoxin and negatively correlated with serum IL-
10. The Lachnospiraceae_NK4A136_group was positively correlated with serum IL-10,
and Clostridium_sensu_stricto_1 was significantly negatively correlated with serum zon-
ulin and ROS levels. The Ruminococcaceae_V9D2013_group was positively correlated
with serum zonulin and ROS and negatively correlated with serum IL-10, and the
Eubacterium_fissicatena_group was positively correlated with serum zonulin, ROS, TNF-a,
and endotoxin, and fecal IL-6. The Norank_f_2534-18b5_gut_group was significantly posi-
tively correlated with fecal IL-6 and negatively correlated with fecal IL-10. There were sig-
nificant positive correlations between unclassified_f_lachnospiraceae and serum zonulin

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and ROS. In addition, the concentrations of acetic acid and butyric acid were positively
correlated with the anti-inflammatory bacteria of the Lachnospiraceae_NK4A136_group
and negatively correlated with the inflammatory bacterium, Terrisporobacter.
In addition, a series of correlation analyses between gut permeability, gut or sys-
temic inflammatory responses, and metabolite markers (Fig. 5B) in sows revealed that
serum ROS concentration was positively correlated with serum IL-6, TNF-a, endotoxin,
and fecal endotoxin and negatively correlated with serum IL-10. The serum TNF-a con-
centration was positively correlated with serum IL-6, and serum endotoxin concentra-
tion was positively correlated with serum IL-6 and TNF-a levels. The concentration of
fecal endotoxin was positively correlated with serum zonulin, endotoxin, IL-6, and TNF-
a. The concentration of acetic acid in feces was positively correlated with that of propi-
onic acid, while butyric acid concentration in feces was negatively correlated with ROS
and IL-6 in serum and positively correlated with acetic acid and propionic acid in feces.
The concentration of total SCFAs in feces was positively correlated with those of acetic
acid, propionic acid, and butyric acid.

TABLE 5 Correlation between inflammatory factors of piglets and sows and weaning weight (L18d)
Correlation (sow group)a

Piglet group Serum lipocalin-2 Serum IL-6 Serum TNF-a Serum IL-10 Fecal IL-6 Fecal TNF-a Fecal IL-10 Serum ROS 21d wt
Serum IL-10 –0.547 –0.698 –0.589 0.836* –0.809 –0.885* 0.799 –0.630 0.324
Serum TNF-a 0.705 0.887* 0.877* –0.691 0.822* 0.750 –0.693 0.832* –0.866*
Serum IL-6 0.951** 0.910* 0.900* 20.627 0.69 0.712 20.735 0.825* 20.730
Serum ROS 0.537 0.719 0.661 20.467 0.635 0.517 20.605 0.746 20.662
aThedata were evaluated by Spearman correlation analysis of the Euclidean distance. *, Mean significant correlation (P , 0.05); **, mean extremely significant correlation
(P , 0.01).

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Liu et al.

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FIG 5 Association and model predictive analysis. (A) Correlation between gut microbiota and host markers
by Spearman correlation analysis. (B) Correlation among host markers by Spearman correlation analysis. *,
0.01 , P # 0.05; **, 0.001 , P # 0.01; ***, P # 0.001. Red indicates a positive correlation; blue indicates a
negative correlation.

DISCUSSION
The immune health status of reproductive sows directly affects overall pig produc-
tivity (3). During pregnancy, sow metabolism is enhanced, which manifests as an
increase in appetite, digestive capacity, weight gain, and storage of numerous
nutrients, to meet the requirements of fetal development. In the later stages of preg-
nancy, in addition to dietary energy, sows cease fat deposition and mobilize stored fat
and energy during pregnancy, which is transferred to the mammary gland for milk syn-
thesis. The metabolism and immune changes in sows during pregnancy and lactation
affect the development and growth of fetuses, and disruption of these adaptive
changes may lead to premature birth or even abortion (4). According to statistical anal-
yses, in commercial genetic lines, the prebirth loss of piglets is around 30 to 50% (29).
Therefore, it is crucial to reduce inflammatory responses in sows during mid and late
pregnancy and lactation and to ensure that the normal metabolic immune changes
occur in the sow reproductive cycle.
Some studies have shown that dietary fiber supplementation not only promotes

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Gut Microbiota and Inflammation in Sows and Piglets

satiety in sows but also improves sow feed intake during lactation (28, 30, 31); how-
ever, the effects of dietary fiber supplementation from different sources are inconsis-
tent and may be closely related to the physicochemical properties and fermentability
of dietary fiber (20, 27, 28, 32, 33). Here, we found that the addition of different fiber
sources to the diet of sows in mid and late gestation affected the performance of both
sows and piglets, with AM in particular significantly reducing the IUGR rate, increasing
feed intake during lactation, and improving sow and piglet performance. AM is rich in
insoluble fiber but also contains a small amount of soluble fiber, which can be fer-
mented in the foregut segment, while the insoluble fiber can be slowly fermented in
the hindgut, and has beneficial effects throughout the intestine (34). Dietary fiber can
prevent and treat gut inflammation induced by a high-carbohydrate and low-fiber
western diet in mice by restoring the damaged gut mucous layer (35). In the case of
long-term or indirect dietary fiber deficiency, the gut microbiota resort to the use of
mucosal glycoprotein secreted by the host as a nutrition source, leading to the erosion
of the mucosal barrier. This results in increased pathogenic bacterial invasion of the
mucosa and aggravation of physiological inflammatory responses (36). Prefeeding
mice with inulin can reduce the gut inflammatory response and Smad7 expression af-
ter infection with Citrobacter rodentium and promote host protective immune
responses by affecting the NF-κB and Smad7 signal transduction pathways (37); how-
ever, longitudinal studies on the dynamic changes in inflammation in sows during mid
and late pregnancy and lactation are insufficient. In a recent study, sows that were in
mid and late gestation and lactating were found to have symptoms of metabolic syn-
drome, mainly characterized by low-level inflammation and metabolic disorder (38).
We assessed the gut permeability, gut and systemic inflammatory responses, and met-
abolic changes in sows fed AM. During the middle and late gestation and lactation
periods in sows fed with AM, we found that three biomarkers of gut permeability (ROS,
endotoxin, and connexin) and markers of inflammation (IL-6, lipocalin-2, and TNF-a)
were decreased, which indicated that AM in the sow’s diet reduced gut permeability
and decreased gut and systemic inflammation. Further, Spearman correlation analysis
showed that there was a significant positive correlation between IL-10 in piglets and

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sows and that TNF-a expression in piglets was positively correlated with IL-6 and TNF-
a expression in sows and negatively correlated with piglet weaning body weight.
Moreover, sow feed intake during lactation was negatively correlated with lipocalin-2,
IL-6, TNF-a, and ROS and significantly positively correlated with IL-10. These findings
suggest that the improvements in performance observed in sows fed with an AM diet
and their piglets are related to the alleviation of gut or systemic inflammatory
responses and the improvement of physical health. Intrauterine developmental retar-
dation remains a major problem in pig production because the associated low birth
weight leads to high preweaning morbidity and mortality and permanent growth and
developmental retardation. Improving the nutritional status of sows in the middle and
late stages of pregnancy can effectively enhance the uniformity of embryos, thereby
reducing changes in embryo development at the placenta stage and intraluminal fetal
weight variation in the later stages of pregnancy (21, 39). Therefore, providing a bal-
anced diet for sows, ensuring normal metabolic immune changes during the reproduc-
tive cycle, and reducing physiological inflammatory responses, particularly in the mid-
dle and late stages of pregnancy, are important for ensuring proper placental nutrient
transport and, ultimately, improving piglet uniformity. In the present study, Spearman
correlation analysis showed that IUGR was positively correlated with lipocalin-2, IL-6,
and TNF-a in sows and significantly negatively correlated with IL-10. Our findings sup-
port the hypothesis that intake of AM in the middle and late gestation period in sows
reduces the occurrence of IUGR by alleviating maternal gut or systemic inflammatory
responses and metabolic disorder.
In addition, increasing numbers of studies have found that gut microbiota are
related to gut permeability and inflammation (40). Changes in the structural compo-
nent of the diet, as the main energy source for gut microbiota, is an effective means of

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Liu et al.

adjusting gut bacteria. Dietary fiber can not only regulate gut microbiota composition
but also adjust microbial metabolites, including SCFAs, etc. Further, it can improve gut
health and influence metabolism and animal behavior (41, 42). Zhao et al. reported
that high dietary fiber could enrich 15 SCFAs produced by bacteria in the gut, stimu-
late SCFA production, improve the gut environment, reduce gut pH, increase butyrate
concentration, competitively inhibit other “harmful bacterial,” and reduce the produc-
tion of harmful metabolites (such as indoles and hydrogen sulfide) and thus build a
healthier gut environment (43). Patients with irritable bowel syndrome have abnormal
gut microbiota due to insufficient intake of SCFAs by gut epithelial cells, and the distri-
bution of tight-junction proteins is directly affected, resulting in thinning of the gut
microbiota, increased gut permeability, and decreased protective effects (44, 45). It has
been suggested that dietary fiber-mediated changes in the gut microbiota and their
metabolites may have important roles in maintaining a gut microecological balance and
ensuring gut health. Our previous study (46) found that adding 5% AM to piglet diet
could inhibit harmful bacteria, such as Mycoplasma and Helicobacter in piglet guts, and
promote the proliferation of beneficial bacteria, such as Paenibacillus, Lactococcus,
Enterococcus, and Faecalibacterium. Compared to the nonpregnant period, the physio-
logical metabolic processes and immune systems of sows exhibit various changes in
pregnancy to meet their physiological needs during this period. The gut microbiota
changes significantly in different periods of pregnancy, which influences host metabolic
processes, and the changes are related to the metabolic characteristics and immune sys-
tem responses specific to the pregnancy period (47). Here, LEfSe analysis showed that
the addition of AM to sow diets in mid and late pregnancy significantly increased the
relative abundances of anti-inflammatory bacteria (Prevotellaceae_NK3B31_group,
norank_f_p_2534_18B5_gut_group, Lachnospiraceae_NK4A136_group, and g_Clostridium_
sensu_stricto_1) and decreased the relative abundance of proinflammatory bacteria
(Terrisporobacter, Desulfovibrio, Helicobacter, Eubacterium_fissicatena_group, and
Erysipelotrichaceae_UCG_004). Previous studies have found that colonization of
Clostridium_sensu_stricto_1 and other bacteria can promote the aggregation of CD41
regulatory T cells in the colon of sterile mice and improve the level of transforming

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growth factor b , while early oral administration of Clostridium microbiota can
increase resistance to colitis and systemic immunoglobulin. Colonization with
Clostridium can also increase the colonization resistance of infant gut microbiota, hin-
dering colonization of pathogenic bacteria (48); however, the colitis that may be
caused by Terrisporobacter could promote gut microbiota malnutrition in animals
(49). Erysipelototrichaeae and other bacteria can promote inflammation in patients
with inflammatory bowel disease (50). Helicobacter is the main cause of chronic active
gastritis and peptic ulcer (51). In addition, we found that the SCFA content in feces of
sows fed with AM had an increasing trend, particularly butyrate at L4d, was significantly
higher than the control. Previous studies have shown that SCFAs can promote the integ-
rity of IL-10 epithelial cells and maintain gut homeostasis by inducing the GPR and
NLRP3 inflammatory pathway (52). Further, butyrate can increase the anti-inflammatory
ability of macrophages and dendritic cells by activating GPR109A, promoting regulatory
T-cell differentiation, increasing the expression of the anti-inflammatory factor IL-10, and
reducing the levels of the inflammatory factors IL-6 and IL-17 (53). Butyrate and propio-
nate can reduce the likelihood of inflammatory bowel disease or colorectal cancer by in-
hibiting the differentiation of regulatory T cells induced by HDACs, maintaining the gut
barrier, and controlling gut inflammation (54, 55). Our findings suggest that the addition
of AM to the diet of sows in the mid and late gestation period can regulate gut
microbiota and SCFA generation, thus improving sow gut health. Spearman correla-
tion analysis further revealed that anti-inflammatory bacterial groups were positively
correlated with anti-inflammatory factors, whereas proinflammatory bacterial groups
were negatively correlated with proinflammatory factors. Moreover, the concentrations of
acetic acid and butyric acid were positively correlated with anti-inflammatory bacteria of
the Lachnospiraceae_NK4A136_group but negatively correlated with anti-inflammatory

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Gut Microbiota and Inflammation in Sows and Piglets

FIG 6 Systematic analysis of the effects of alfalfa meal diet on growth performance, inflammatory indexes, gut microbiota, and SCFAs of sows and piglets.

Terrisporobacter bacteria. Serum ROS levels were significantly positively correlated with
proinflammatory factors and endotoxins but negatively correlated with anti-inflamma-
tory factors, and there was a significant positive correlation between serum endo-
toxin and proinflammatory factors, as well as a significant positive correlation

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between fecal endotoxin concentration and zonulin. Furthermore, BA was signifi-
cantly negatively correlated with proinflammatory factors, such as serum ROS and se-
rum IL-6. Therefore, we conclude that the addition of AM in the middle and late ges-
tation period of sows may improve disordered gut microbiota and decreased SCFAs
generation, thus relieving the gut and systemic inflammatory response and promot-
ing the healthy growth of sows.
Using combined correlation analysis of growth performance, inflammatory indices,
gut microbiota, and SCFAs in sows and piglets (Fig. 6), we propose that excessive ROS
produced by overactive metabolic processes during late pregnancy and lactation will
lead to increased endotoxin levels, disordered gut microbiota, decreased SCFA produc-
tion, and secretion of proinflammatory factors, which will cause local inflammation of
the gut, potential damage of the gut microbial barrier, increased gut permeability,
increased blood endotoxin levels resulting in systemic inflammation, and ultimately,
decreased sow and piglet performance. Supplementation of the diet with AM in mid
and late pregnancy can reverse this process. Specifically, AM can increase the abun-
dance of anti-inflammatory bacteria and reduce gut proinflammatory bacterial abun-
dance by regulating the gut microbiota structure and SCFA production by AM fermen-
tation, which decreases endotoxin and inflammatory factor secretion in the blood,
resulting in reduced physiological inflammatory responses and improved sow perform-
ance, as well as reducing inflammatory responses in suckling piglets, and finally
improving piglet gut health and growth performance. Nevertheless, further research is
needed to elucidate the specific mechanisms underlying the interactions among gut
microbiota, gut permeability, and the inflammatory and metabolic characteristics of
sows and piglets.
In conclusion, we found that the addition of different fiber sources to the diet

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Liu et al.

during mid and late gestation influenced the performance of sows and piglets. In par-
ticular, the addition of AM significantly improved sow and piglet performance and
relieved gut and systemic inflammation. Furthermore, the supplementation with AM
significantly increased the relative abundance of anti-inflammatory bacteria and
decreased that of proinflammatory bacterial. We propose that the improvement in the
performance of sows and piglets can be ascribed to the beneficial effects of AM on gut
microbiota and the SCFA generation, resulting in decreased inflammatory responses
and enhanced physical health in sows and piglets. These findings provide a theoretical
basis and guide for the use of specific fiber sources in the diet of sows to improve gut
health and production performance of sows and piglets. Our data also give insights for
the study of the role of dietary fiber in the gastrointestinal function of human mothers
and infants.

MATERIALS AND METHODS

Ethical approval. All experimental procedures in this study were approved by the Institutional
Animal Ethics Committee of Henan Agricultural University (approval HENAU-2018-015).
Animals, diets, and housing. Based on similar expected dates of confinement and backfat thick-
ness, 48 sows (Large  Landrace) at 60 days of gestation were randomly allocated to the control (CK),
alfalfa meal (AM), beet pulp (BP), and soybean skin (SH) groups. Each treatment included 12 replicated
pens, each of which housed one sow. The preparation period was 7 days, and the test period was
75 days. All pregnant sows were supplied with feed formulated to meet National Research Council 2012
recommendations (56). The detailed ingredient composition and nutrient content of the investigated
diets are presented in Table S1 in the supplemental material. On day 107 of pregnancy, sows were
moved to individual farrowing pens with crates, slatted floors, and heat pads for the piglets. At parturi-
tion, the numbers of stillborn and live-born piglets in each litter were recorded. In the 12 h after farrow-
ing, the litter size and individual piglet birth weights were measured. When possible, litter sizes were
adjusted to 11 to 12 piglets, by adding or removing piglets within each dietary group without changing
the mean litter birth weight. Lactating sows all consumed the same diet. Both sows and piglets had free
access to water. The sow back fat thickness during the reproductive cycle and reproductive perform-
ance, as well as the growth performance of piglets, was recorded.
Sample collection. At 100 days of gestation and at 4 and 18 days of lactation, four sows were
selected from each treatment for collection of serum and fecal samples, and piglet blood samples were
also collected at 4 and 18 days of lactation. Serum samples (5 ml) were collected in heparinized tubes
from the vena jugularis of sows and piglets, with a minimal amount of stress. Plasma samples were then

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obtained by centrifuging the serum samples at 3,000  g at 4°C for 10 min and stored at –80°C until anal-
ysis. Fresh fecal samples were collected individually from the pigs using sterile 20-ml centrifuge tubes
and then stored at –80°C until analysis. According the performance indicators of sows, the optimal treat-
ment was selected for the measurement of inflammatory factors, SCFA levels, and 16S rRNA gene
sequencing.
Measurement of inflammatory factors. IL-6, IL-10, TNF-a, endotoxin, zonulin, lipocalin-2, and ROS
were measured in sow serum samples, and IL-6, IL-10, TNF-a, and endotoxin in sow stool samples. IL-6, IL-
10, TNF-a, endotoxin, and ROS were also measured in piglet serum samples. Inflammatory factors were
evaluated using enzyme-linked immunosorbent assay technology (Nanjing Jiancheng Bioengineering
Institute, Nanjing, China). All procedures were performed in duplicate.
Determination of SCFAs levels. Gas chromatography (GC) performed as described by Liu et al. (46)
was used to determine the SCFA levels of in stool samples. The samples were analyzed on an HP-88 col-
umn (100-m length, 0.25-mm diameter, and 0.2-m m film thickness from the producer) and separated
using a TRACE 1310 GC with a flame ionization detector. The temperature program was as follows: 70°C
for 1 min, followed by an increase to 180°C held at 25°C for 1 min, an increase to 200°C held at 10°C for
1 min, an increase to 220°C held at 2°C for 10 min, and finally an increase to 240°C held at 20°C for 6
min. The sample was run with a split ratio of 20:1 and a column flow rate of 1.3 ml/min. Hydrogen is
used as a carrier gas. The injector temperature is 270°C, and the detector temperature is 290°C.
DNA extraction and 16S rRNA gene sequencing. Microbial DNA was extracted from feces samples
by using an E.Z.N.A. soil DNA kit (Omega Bio-Tek, Norcross, GA) according to the manufacturer’s proto-
cols. The final DNA concentration and purity were determined by using a NanoDrop 2000 UV-vis spec-
trophotometer (Thermo Scientific, Wilmington, DE), and the DNA quality was checked by 1% agarose
gel electrophoresis. The V3-V4 hypervariable regions of the bacterial 16S rRNA gene were amplified
using the primers 338F (59-ACTCCTACGGGAGGCAGCAG-39) and 806R (59-GGACTACHVGGGTWTCTAAT-
39) by PCR (GeneAmp 9700; ABI) (57), with the following program: 3 min of denaturation at 95°C; 27
cycles of 30 s at 95°C, 30 s of annealing at 55°C, and 45 s of elongation at 72°C; and a final extension at
72°C for 10 min. PCRs were performed in triplicate, with each 20-m l reaction mixture containing 4 m l of
5 FastPfu buffer, 2 m l of 2.5 mM deoxynucleoside triphosphates, 0.8 m l of each primer (5 m M), 0.4 m l of
FastPfu polymerase, and 10 ng of template DNA. The resulting PCR products were extracted from 2%
agarose gels, further purified using the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City,
CA), and quantified using a QuantiFluor-ST instrument (Promega) according to the manufacturers’ proto-
cols. Purified amplicons were pooled in equimolar amounts and subjected to paired-end sequencing

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Gut Microbiota and Inflammation in Sows and Piglets

(2  300 bp) on an Illumina MiSeq platform (Illumina, San Diego, CA), according to standard protocols,
by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).
Bioinformatics analysis of sequencing data. Raw fastq files were demultiplexed, quality filtered
using Trimmomatic, and merged using FLASH, according to the following criteria: (i) reads were trun-
cated at any site receiving an average quality score of ,20 over a 50-bp sliding window; (ii) primers
were exactly matched, allowing 2-nucleotide mismatching, and reads containing ambiguous bases
removed; and (iii) sequences whose overlap was longer than 10 bp were merged, according to their
overlap sequence. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using
UPARSE (v7.1 [http://drive5.com/uparse/]), and chimeric sequences identified and removed using
UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed using the RDP Classifier algo-
rithm (http://rdp.cme.msu.edu/) against the Silva (SSU128) 16S rRNA database, with a 70% confidence
threshold. Sample biodiversity was calculated using the ACE, Chao1, and Shannon indices by applying a
Wilcoxon rank sum test. Beta-diversity measures dependent on Bray-Curtis and unweighted-UniFrac dis-
tance values were calculated using mothur. LEfSe analysis was conducted to identify bacterial taxa dif-
ferentially represented between different groups at the phylum to genus taxonomy level (biomarkers)
by one-against-all (less strict). To determine the effect of microbiota interacting with Apparent perform-
ance, redundancy analysis (RDA) was performed at the genus level using the R language vegan packet
on Spearman correlation analysis (RDA 2014).
Statistical analysis. Statistical analyses were performed using SPSS 20.0 software (IBM, New York,
NY). Data were evaluated by one-way ANOVA, and the differences between means assessed using
Duncan’s test. A P value of ,0.05 was considered statistically significant. The data were evaluated by
Spearman correlation analysis of the Euclidean distance.
Data availability. Raw reads were deposited into the NCBI Sequence Read Archive database under
accession number SRP268238.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
FIG S1, JPG file, 0.9 MB.
TABLE S1, DOCX file, 0.03 MB.

ACKNOWLEDGMENTS
Financial support for this research was provided by the Earmarked Fund for
Modern Agro-industry Technology Research System of China (CARS-34) and the
Science and Technology Research Key Project of Education Department of Henan
Province (19B230012).

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B.L. and X.Z. performed experiments and analyzed data. Y.C. participated in the data
collection. W.W., H.L., Z.L., and Z.G. assisted with animal experimentation. S.M., D.L., and
C.W. provided advice in design and performance of experiments. B.L. wrote the
manuscript draft. Y.S. supervised the study. All authors read and approved the final
manuscript.

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