Diets effect on growth performance of weaned piglets
As shown in Table 3, piglets fed with ChB diet gained a greater body weight and a significantly higher ADG than those fed control diet during the post-weaning period (BW:26.03 ± 0.74 vs 24.51± 0.91 kg; ADG: 638.4± 16.1 vs 580.8 ±22.9 g/d), and a visibly lower F/G (1.176± 0.034 vs 1.27±0.016). Piglets fed the choline diet tended to have a same variable trend as ChB diet for body weight and ADG, but without statistics. Whereas choline group had a significantly lower F/G than those fed control diet. No differences in body weight, ADG and F/G were observed between bile acids group and control group. Furthermore, there was no difference in ADFI among the four groups.
Item
|
Control
|
Choline
|
BAs
|
ChB
|
Initial BW kg
|
8.24± 0.44
|
8.22 ± 0.43
|
8.21 ± 0.44
|
8.17 ± 0.42
|
Final BW kg
|
24.51± 0.91
|
25.88 ± 0.82
|
24.40 ± 0.69
|
26.03 ± 0.74
|
ADG, g
|
580.8 ±22.9b
|
630.4 ± 18.2ab
|
576.9± 27.4b
|
638.4± 16.1a
|
ADFI, g
|
771.4±27.01
|
739± 20.05
|
706.0± 19.16
|
747.3± 11.15
|
F/G
|
1.27±0.016a
|
1.18± 0.038b
|
1.23± 0.048a
|
1.17± 0.034b
|
Diarrhea index %
|
2.09± 1.03
|
0
|
0
|
0
|
Data presented as means±SEM, and different superscripts in the same row are significantly different (p < 0.05). N=32. BAs, bile acids; ChB, choline and bile acids mixture.
|
Table 3
Diet effects on the growth performance in weaned piglets.
Diet effect on biochemical indexes related to fats digestion in intestinal digesta of weaned piglets
As shown in Table 4, piglets fed ChB displayed a notably higher lipase activity in jejunal digesta, and significantly higher level of total bile acids in both jejunal and ileal digesta than those fed control diet. However, there were no significant differences in lipase activity and total bile acids concentration between choline and control group or bile acids and control group. In addition, both choline and ChB supplementation significantly increased the NEFA content in the jejunal digesta compared to control diet. No difference for NEFA content in the jejunal digesta was found between bile acids group and control group. However, bile acids decreased NEFA content in the ileal digesta compared with control diet. Furthermore, choline, bile acids as well as ChB diet significantly decreased TG concentration in the jejunal digesta compared with control diet. Choline group or bile acids group also showed a profound reduction of TG concentration in the ileal digesta, when compared to the control group. Besides, both choline diet and bile acids diet significantly reduced the T-CHO level in the duodenal digesta compared to control diet. T-CHO level in the duodenal digesta of ChB group tend to be lower than the control group, but without statistics. In the jejunal digesta, bile acids diet markedly decreased the level of T-CHO, as compared with control diet.
Items
|
Control
|
Choline
|
BAs
|
ChB
|
Lipase activity in intestinal digesta (U/gprot)
|
Duodenum
|
43.14± 12.55
|
34.23 ± 6.65
|
31.51±2.51
|
42.93± 22.06
|
Jejunum
|
115.58± 0.18.33b
|
189.04 ± 50.76ab
|
139.57 ± 25.94ab
|
221.13 ± 0.43a
|
Ileum
|
31.66± 5.97
|
52.33± 8.60
|
35.20 ± 6.22
|
51.00 ± 3.92
|
Bile acid content in intestinal digesta (umol/gprot)
|
Duodenum
|
626.58± 93.90
|
517.62±49.46
|
528.41±130.05
|
529.65± 6.60
|
Jejunum
|
457.51± 77.44b
|
506.32±39.40ab
|
505.09±76.11ab
|
647.75± 76.11a
|
Ileum
|
15.36± 2.43b
|
29.21±7.95b
|
29.85±10.43b
|
175.99± 24.83a
|
NEFA content in intestinal digesta (umol/gprot)
|
Duodenum
|
0.63± 0.09
|
0.99±0.25
|
0.93±0.09
|
1.03± 0.33
|
Jejunum
|
1.12± 0.25b
|
1.89±0.33a
|
0.87±0.18b
|
1.90± 0.16a
|
Ileum
|
0.35± 0.12a
|
0.19±0.027ab
|
0.13±0.01b
|
0.38± 0.09a
|
TG content in intestinal digesta (mmol/gprot)
|
Duodenum
|
2.17± 0.19
|
2.30±0.31
|
2.40±0.56
|
2.31± 0.66
|
Jejunum
|
6.34±1.14a
|
3.38±0.38b
|
2.60±0.36b
|
2.97± 0.41b
|
Ileum
|
1.52±0.15a
|
1.09±0.12b
|
1.01±0.086b
|
1.29± 0.15ab
|
T-CHO content in intestinal digesta (mmol/gprot)
|
Duodenum
|
2.08±0.62a
|
0.94±0.17b
|
0.95±0.12b
|
1.23± 0.32ab
|
Jejunum
|
2.18±0.30a
|
1.87±0.29a
|
0.36±0.17b
|
1.53± 0.33a
|
Ileum
|
1.35±0.29
|
2.15±0.67
|
1.54±0.19
|
1.27± 0.42
|
Data presented as means±SEM, and different superscripts in the same row are significantly different (p < 0.05). N=8. BAs, bile acids; ChB, choline and bile acids mixture.
|
Table 4
Biochemical indexes related to fats digestion in intestinal digesta of weaned piglets.
Diets induced changes in the composition of colonic microbiota of weaned piglets
A total of 16704 operational taxonomic units (OTUs) (522 OTUs per sample) from 32 samples were identified and for further analysis. Venn analysis of OTUs showed that 151, 45, 55 and 27 unique OTUs were identified in control group, choline group, bile acids group and ChB group, respectively, and 666 OTUs were shared among the four groups (Figure 1a). The diversity and richness of the colonic digesta microbiota were present in Table 5. ChB supplementation caused reduction in richness and diversity indices compared with control diet, as reflected by the reduction in Shannon, Simpson and ACE index with statistical differences. Beta-diversity analysis showed clustering of samples according to diets. In particular, the PCA results showed that ChB group and choline group were separated from control group (Figure 1b). Diet-induced changes in bacterial composition were clearly visible at the genus level. Lactobacillus and Faecalibacterium were considerably more abundant in piglets fed ChB than those in control group. In addition, the relative abundances of unidentified-Clostridiales, Parabacteroides and Unidentified-Ruminococcacace were decreased by ChB, as compared with the control diet. Piglets fed choline showed a decreased abundance of Parabacteroides compared to those fed control diet (Figure 1c, d). Bile acids increased the abundance of Bacteroides compared with control diet, with a decrease in the abundance of Parabacteroides. Besides, choline or bile acids diet can slightly increase the relative abundances of Lactobacillus, and decrease the relative abundances of Unidentified-Ruminococcacace, but without statistics significant. Furthermore, LEfSe analysis (LDA score >4; Figure1e) showed significant distinguishing bacteria with class Bacilli, order Lactobacillales, family Lactobacillaceae, genus Lactobacillus in the ChB group, with family unidentified Clostridiales and Bacteroidaceae, genera Bacteroides and Actinobacillus in the bile acid group, and with the family Muribaculaceae in the control group when compared with the other three groups (Figure 1e).
Items
|
Control
|
Choline
|
BAs
|
ChB
|
Diversity indices
|
Shan
|
6.04± 0.24a
|
5.41 ± 0.13b
|
5.66±0.21ab
|
5.32± 0.17b
|
Simpson
|
0.95± 0.0.015a
|
0.92 ± 0.008ab
|
0.93 ±0.12ab
|
0.91 ± 0.012b
|
Richness
|
Chao1
|
652.30± 44.93
|
585.22±11.86
|
665.83±22.42
|
589.88± 33.56
|
ACE
|
666.28±47.45a
|
590.51±9.91ab
|
663.41±18.38ab
|
578.29± 23.71b
|
Data presented as means± SEM, and different superscripts in the same row are significantly different (p < 0.05). N=8. BAs, bile acids; ChB, choline and bile acids mixture.
|
Table 5
Summary statistic of colonic digesta bacterial community at the 3% dissimilarity level.
Diet effect on SCFA production in weaned piglets
No significant differences in propionate level among four groups were found (Figure 2). Choline induced an increase in the level of acetate compared with control group. Compared to piglets fed control diet, those fed ChB diet showed a profound elevation of the concentration of butyrate. However, there was not different in the concentration of butyrate among the other three groups.
Diet effect on bile acid profiles level in weaned piglets
As shown in Figure 3, in the colonic digesta, levels of primary bile acids including chenodeoxycholic acid (CDCA) and 3β-ursodeoxycholic acid (3βUDCA), and secondary bile acid including hyodeoxycholic acid (HDCA) and ursodeoxycholic acid (UDCA) were significantly decreased in ChB group, as compared with the control group. Choline supplementation decreased the relative levels of 23-norcholic acid (23-NCA), 3βUDCA, HDCA and UDCA compared with control group. Piglets fed bile acids displayed significantly higher levels of β- muricholic acid (MCA) and α-MCA compared to those fed control diet.
Diet effect on the proteomic profile related to innate immune response in jejunal mucosa of weaned piglets
As shown in Supplementary figure 1a, b, c and d, volcano plot was used to illustrate the differentially expressed proteins (DEPs) expression patterns, from the volcano plot we found that more DEPs were identified in the jejunal mucosa sample of weaned piglets fed ChB diet. Compared with the control group, there were 35 upregulated proteins and 45 downregulated proteins in the choline group, 84 upregulated proteins and 46 downregulated proteins in bile acid group, 132 upregulated proteins and 84 downregulated proteins in the ChB group, respectively. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was used to collect the DEPs function information of the jejunal mucosa samples from the four groups. Particularly, DEPs involved in immune system were payed close attention and described utilizing the heatmap analyses. As shown in Figure 4a, b, c and Table 6, there were 11,10 and19 DEPs related to innate immune system in the choline group, bile acid group and ChB group, respectively, as compared with the control group. In addition, the DEPs accumulated to NF-κB, Toll-like receptor and NOD-like receptor signaling pathway, which were highly involved in inflammation, were analyzed. Compared to the control diet, choline diet upregulated Ig-like domain-containing protein and Interleukin-18, while downregulated IgM and tripartite motif-containing protein. Piglets fed bile acids had significant higher levels of Ig-like domain-containing protein and tyrosine-protein kinase, but lower levels of mitogen-activated protein kinase 7, IgG heavy chain and thioredoxin-interacting protein isoform 1 than those fed control diet. The amount of protein kinase domain-containing protein, Ig-like domain-containing protein (FCGRT), PPM-type phosphatase domain-containing protein (PDP2), Integrin beta, ANK_REP_REGION domain-containing protein and NF-κB inhibitor epsilon were significantly increased in ChB group, while IgG heavy chain, IgM, probable ATP-dependent RNA helicase DDX58(DDX58), MyD88 and BCL10 immune signaling adaptor (BCL10) were markedly down-regulated, as compared with the control diet.
|
Protein ID
|
Description
|
Gene
|
FC
|
P value
|
choline
|
A0A4X1SUP3
|
Ig-like domain-containing protein
|
1.80
|
0.02
|
|
O77947
|
MHC class I antigen 2 (Fragment)
|
1.30
|
0.01
|
|
A0A287AH22
|
Membrane cofactor protein
|
CD46
|
1.28
|
0.03
|
|
A0A2C9F3D6
|
Interleukin-18
|
IL18
|
1.21
|
0.03
|
|
A0A1P8VJR2
|
Aminopeptidase
|
APN
|
1.21
|
0.03
|
|
A0A480MNY7
|
Platelet endothelial cell adhesion molecule isoform X1
|
0.82
|
0.03
|
|
A0A480Z8U9
|
Guanine nucleotide exchange factor VAV2 isoform 2 (Fragment)
|
0.78
|
0.02
|
|
A0A287ADL4
|
Polymeric immunoglobulin receptor
|
PIGR
|
0.69
|
0.02
|
|
A0A480K9S0
|
1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase
|
0.66
|
0.05
|
|
A0A480QMR6
|
IgM
|
IgM
|
0.68
|
0.04
|
|
B6ICV0
|
Tripartite motif-containing protein 15
|
TRIM15
|
0.76
|
0.03
|
BAs
|
A0A4X1VTZ6
|
Protein kinase domain-containing protein
|
MAP2K2
|
1.65
|
0.01
|
|
A0A075B7I5
|
Ig-like domain-containing protein
|
|
1.32
|
0.03
|
|
G0KXP9
|
MHC class I antigen
|
SLA-3
|
1.30
|
0.05
|
|
A0A4X1W3V2
|
Tyrosine-protein kinase
|
LOC100739325
|
1.29
|
0.04
|
|
A0A1P8VJR2
|
Aminopeptidase
|
APN
|
1.25
|
0.02
|
|
A0A480UWI6
|
Plasma protease C1 inhibitor
|
0.69
|
0.03
|
|
A0A4X1SK84
|
Neutrophil cytosolic factor 1
|
NCF1
|
0.80
|
0.02
|
|
B0LXP5
|
Mitogen-activated protein kinase 7
|
TAK1b
|
0.82
|
0.00
|
|
L8AXK8
|
IgG heavy chain
|
IGHG
|
0.78
|
0.01
|
|
A0A480K2Q0
|
Thioredoxin-interacting protein isoform 1
|
0.63
|
0.03
|
ChB
|
A0A4X1VTZ6
|
Protein kinase domain-containing protein
|
MAP2K2
|
1.68
|
0.01
|
|
A0A4X1VYT4
|
Ig-like domain-containing protein
|
FCGRT
|
1.56
|
0.04
|
|
A0A4X1ULK6
|
PPM-type phosphatase domain-containing protein
|
PDP2
|
1.28
|
0.03
|
|
A0A4X1T9Z3
|
Integrin beta
|
ITGB3
|
1.27
|
0.01
|
|
K7GSL6
|
ANK_REP_REGION domain-containing protein
|
BCL3
|
1.25
|
0.04
|
|
A0A4X1SNA9
|
Protein kinase domain-containing protein
|
MAP2K4
|
1.22
|
0.04
|
|
A0A4X1V587
|
NFKB inhibitor epsilon
|
1.22
|
0.05
|
|
A0A286ZZN4
|
Protein kinase domain-containing protein
|
GSK3A
|
0.82
|
0.05
|
|
A0A480Z8U9
|
Guanine nucleotide exchange factor VAV2 isoform 2 (Fragment)
|
0.82
|
0.03
|
|
A0A286ZT79
|
RPOLD domain-containing protein
|
POLR1C
|
0.78
|
0.03
|
|
A0A0A0MY60
|
Signal transducer and activator of transcription
|
STAT5A
|
0.77
|
0.01
|
|
A0A4X1W2Z9
|
C1q domain-containing protein
|
C1QB
|
0.74
|
0.01
|
|
A0A287ADL4
|
Polymeric immunoglobulin receptor
|
PIGR
|
0.72
|
0.03
|
|
F1RX35
|
Fibrinogen C-terminal domain-containing protein
|
FGG
|
0.71
|
0.03
|
|
L8B0R9
|
IgG heavy chain
|
IGHG
|
0.76
|
0.00
|
|
A0A480QMR6
|
IgM
|
|
0.58
|
0.02
|
|
A0A4X1UKT2
|
Probable ATP-dependent RNA helicase DDX58
|
DDX58
|
0.70
|
0.03
|
|
A0A140TAK4
|
Myeloid differentiation primary response protein MyD88
|
MYD88
|
0.79
|
0.03
|
|
A0A4X1UEV0
|
BCL10 immune signaling adaptor
|
BCL10
|
0.61
|
0.01
|
N=8. BAs, bile acids; ChB, choline and bile acids mixture.
|
Table 6
Differentially expressed proteins in the pathway of immune system compared with control diet fed weaned piglets.
Diet effect on the intestinal mucosal barrier function of the weaned piglets
As shown in Figure 5, there was no significant effect of diet treatments on the abundance of claudin-1 and occludin transcripts in the intestine. However, ChB supplementation profoundly elevated the abundance of IL-10 transcript, while significantly decreased MyD88, TLR4 and TNF-α genes expression in the duodenal mucosa compared to the control diet, the transcript level of NF-κBp65 tended to have the same variable trend, but without no statistics. In the jejunal mucosa, ChB diet significantly increased the expression of mucin2 gene, while markedly decreased abundance of MyD88, TLR4, TNF-α and NF-κBp65 transcripts. Piglets fed ChB showed a higher amount of ileal IL-10 gene expression. Choline supplementation induced a visibly higher abundance of IL-10 transcript and lower abundance of TNF-α transcript in the duodenal mucosa, lower abundance of TLR4, TNF-α and NF-κBp65 transcripts in the jejunal mucosa, when compared with those fed control diet. Additionally, bile acids supplementation significantly decreased the gene expressions of TNF-α and MyD88 in the duodenal mucosa, and markedly decreased the levels of TLR4, TNF-α and NF-κBp65 transcripts in the jejunal mucosa, but increased the abundance of IL-10 transcript in the ileal mucosa, as compared with the control diet.
Diet effect on the expression of FXR and TGR5 genes
No difference in TGR5 gene expression in the ileal and colonic mucosa of weaned piglets was observed among four groups. However, the expression of FXR gene in the colonic mucosa was increased in choline group and ChB group compared to control group. However, no difference for this expression in ileal mucosa among the four groups was observed (Figure 6).
Correlation analysis between gut microbiota, bacterial metabolites, ADG and intestinal genes expression
A Pearson correlation analysis was used to investigate the associations between genes expression and the abundance of the main microbial genera and their metabolites (Figure 7). The results revealed that the expression of TLR4 transcript was significantly negatively correlated with the abundance of Lactobacillus, while positively related to CDCA. MyD88 genes expression showed positive correlations with HDCA and UDCA, while it was negatively associated with butyrate. The abundance of TNF-a gene was positively correlated with level of UDCA. The abundance of NF-κBp65 transcript had a negative correlation with the relative abundance of Lactobacillus. FXR gene expression was significantly correlated with the abundance of Lactobacillus, while negatively correlated with the level of 23-NCA, HDCA and UDCA as well as the relative abundance of Parabacteroides and Unidentified ruminococcaceae. The abundance of IL-10 transcript showed positive association with acetate level and the relative abundance of Lactobacillus and Faecalibacterium, while showed negative correlation with HDCA, UDCA and Parabacteroides. In addition, ADG was positively correlated with the relative abundance of Lactobacillus and butyrate level.