Alpha And Beta Diversity Analysis
The alpha diversity indices (Chao1, observed species, Shannon, and Simpson) are shown in Table 1. The richness of the microbiota in the feces from the SC group was significantly lower than that in the CON group at the age of 16 and 35 days, as measured by the Chao1 and observed species indices (P < 0.001), while no significant difference was observed in the CON and SC groups at 42 and 63 days (P > 0.05). When compared to the CON group, the Shannon index in the SC group was significantly lower at 16 days of age (P = 0.018), but showed no significant difference at 35, 42, and 63 days of age (P > 0.05). However, the Simpson index was significantly decreased in SC group at 16, 42, and 63 days compared with the CON group (P < 0.05), but showed no difference at 35 days of age (P > 0.05). Based on Bray-Curtis distance and weighted Unifrac distances, the PCoA plot justified that the microbiota of CON and SC groups were relatively separated at any of the sampled time-points (P < 0.001) (Fig. 2).
Table 1
Effects of social contact on alpha diversity of fecal microbiota in piglets
Items
|
n
|
Chao1
|
Observed species
|
Shannon
|
Simpson
|
16 d
|
CON
|
20
|
415.75 ± 119.88
|
380.50 ± 103.24
|
5.96 ± 0.84
|
0.95 ± 0.05
|
SC
|
20
|
263.71 ± 101.79
|
253.50 ± 96.37
|
5.60 ± 0.65
|
0.94 ± 0.03
|
P-value
|
-
|
2.9e-0.5***
|
< 0.001***
|
0.018*
|
0.035*
|
35 d
|
CON
|
20
|
728.28 ± 229.27
|
657.45 ± 196.97
|
6.70 ± 1.01
|
0.96 ± 0.05
|
SC
|
20
|
479.89 ± 222.43
|
453.55 ± 191.28
|
6.69 ± 0.96
|
0.97 ± 0.03
|
P-value
|
-
|
0.002**
|
0.004**
|
0.990
|
0.600
|
42 d
|
CON
|
20
|
481.76 ± 141.73
|
449.55 ± 124.44
|
7.39 ± 0.46
|
0.99 ± 0.004
|
SC
|
15
|
426.87 ± 120.52
|
401.60 ± 109.48
|
6.81 ± 0.70
|
0.97 ± 0.03
|
P-value
|
-
|
0.180
|
0.370
|
0.180
|
0.012*
|
63 d
|
CON
|
20
|
452.38 ± 102.45
|
438.55 ± 96.89
|
7.32 ± 0.42
|
0.98 ± 0.01
|
SC
|
16
|
487.41 ± 144.66
|
467.75 ± 131.38
|
7.12 ± 0.57
|
0.97 ± 0.02
|
P-value
|
-
|
0.300
|
0.390
|
0.320
|
0.023*
|
Note. The data are presented as means ± SD. CON, control group; SC, social contact group. No superscript represents no significant difference (P > 0.05), *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively. |
Microbial Composition
The composition of fecal microflora of piglets at the age of 16, 35, 42 and 63 days is shown in Fig. 3. At the phylum level (Fig. 3A), Firmicutes and Bacteroidetes were the dominant phyla in the feces at all time-points, with an abundance of over 80% of the population and even more than 90% after weaning. The abundance of Firmicutes in both groups increased with age except at 42 days of age. The abundance of Firmicutes and Bacteroidetes in the SC group showed no significant difference compared with the CON group at 16 days of age (P > 0.05). However, the abundance of Firmicutes in the SC group was notably higher than that in the CON group at 35, 42 and 63 days of age (P < 0.05), while the abundance of Bacteroidetes in the SC group was notably lower than that in the CON group at 35, 42 and 63 days of age (P < 0.05). The remaining bacteria identified were Actinobacteria, Proteobacteria, Spirochaetes, and Fusobacteria. The abundance of Actinobacteria in the SC group was significantly higher than that in the CON group at 16, 35, and 63 days of age (P < 0.05), as was the proportion of Proteobacteria at 63 days of age (P < 0.05). However, the proportion of Proteobacteria was notably lower in SC pigs at 16 and 35 days of age (P > 0.05). Finally, the abundance of Fusobacteria in the SC group was significantly decreased at 35 days of age compared with that of the CON group (P < 0.05). In general, the phylum-level analysis determined that the group exposed to a social environmental had an increase in the proportion of Firmicutes and Actinobacteria and a decrease in the proportion of Bacteroidetes. Besides, the Firmicutes-to Bacteroidetes ratio in the SC group was significantly higher than that in the CON group at the age of 35, 42, and 63 days (P < 0.05), but was not significantly different at 16 days of age (P > 0.05) (Fig. 4).
At the family level (Fig. 3B), Ruminococcaceae formed the largest proportion of the microbiome at all detection times in both groups. However, Muribaculaceae was the second-largest proportion of bacteria during the suckling period (16 and 35 days of age) in both groups but decreased after weaning (42 and 63 days of age). Meanwhile, Lactobacillaceae and Lachnospiraceae were increased after weaning in both groups, to become the second and third largest proportion of the population, respectively. The proportion of Prevotellaceae in the SC group was significantly decreased than for the CON group at 35, 42, and 63 days of age (P < 0.05), while the proportion of Bacteroidaceae in the SC group was less than that in the CON group at the age of 16, 35, and 63 days (P < 0.05), but increased significantly at 42 days of age (P < 0.05).
At the genus level (Fig. 3C), Muribaculacesae was the most prevalent genus during the suckling period in both groups, while Lactobacillus and Muribaculacesae were the most prevalent genera during the growing period. Meanwhile, the relative abundance of Bacteroides was enriched at the suckling period, but gradually decreased with the age. Conversely, the proportion of Ruminococcaceae_UCG-014, Faecalibacterium and Subdoligranulum were very low during the suckling period, but they continuously increased with the age. In addition, Fig. 5 (A, B, C, and D) showed that the proportion of Lactobacillus in the SC group was significantly increased compared with the CON group at all time-points (P < 0.05), while the abundance of Prevotella in the SC group was significantly decreased over that of the CON group (P < 0.05). Other genera fluctuated, but the proportions of Eubacterium-coprostanoligenes_group, Ruminococcaceae_UCG-002, and Ruminococcaceae_UCG-005 in the SC group were markedly increased compared with the CON group at 63 days (P < 0.05) while the abundance of Alloprevotella significantly decreased in the SC group (P < 0.05).
Functional changes in the fecal microbiota induced by social contact
PICRUSt was applied in this study to investigate the functional and metabolic potentials of the pig fecal microbiota and the possible changes induced by social contact based on the next-generation sequencing reads. According to the results, the most abundant COG families in the feces at 16 and 35 days of age were response regulators including Signal transduction histidine kinase (COG#0642), Site-specific recombinase XerD (COG#4974), Response regulators consisting of a CheY-like receiver domain and a winged-helix DNA-binding domain (COG#0745), AraC-type DNA-binding domain-containing proteins (COG#2207), and Na+-driven multidrug efflux pump (COG#0534) (Additional file 2: Table S2 and Additional file 3: Table S3). At 35 and 42 days of age, the most abundant COG families were response regulators including Signal transduction histidine kinase (COG#0642), Site-specific recombinase XerD (COG#4974), Response regulators consisting of a CheY-like receiver domain and a winged-helix DNA-binding domain (COG#0745), ABC-type multidrug transport system, ATPase and permease components (COG#1132), and Na+-driven multidrug efflux pump (COG#0534) (Additional file 4: Table S4 and Additional file 5: Table S5). Pre- and post-weaning, there was one change in the composition of the COG families. At these time-points, the most abundant COG functional categories (classes) were Signal transduction mechanisms (category T), Defense mechanisms (category V), Transcription (category K), and Replication, recombination, and repair (category L). The social contact had a significant effect on the functional categories of the microbiota in the feces, as shown in Tables 2–5. For example, the abundance of Post-translational modification, protein turnover, and chaperones (category O), carbohydrate transport and metabolism (category G), and coenzyme transport and metabolism (category H) in the SC group were notably different from the CON group (P < 0.05). Categories O and H significantly increased at 16 days of age for SC compared with the CON group (P < 0.05), but were significantly decreased at 35, 42, and 63 days of age (P < 0.05). Conversely, the G category in the SC group significantly decreased at 16 days of age compared to the CON group (P < 0.05), but significantly increased at 35, 42, and 63 days of age (P < 0.05). The social contact implied to have significant effects on some functional categories of microbiota in the feces. Especially in cellular processes and signaling, Functional categories for: the abundance of Cell wall/membrane/envelope biogenesis (category M), Post-translational modification, protein turnover, and chaperones (category O) and Intracellular trafficking, secretion, and vesicular transport (category U) were notably lower in the SC group than in the CON group at 35, 42 and 63 days of age (P < 0.05). Additionally, for metabolism categories, when compared to the CON group, the abundance of Coenzyme transport and metabolism (category H) and Lipid transport and metabolism (category I) were significantly lower in the SC group at 35, 42 and 63 days of age (P < 0.05), while the abundance of Carbohydrate transport and metabolism (G category) was significantly higher (P < 0.05), as was the abundance of Amino acid transport and metabolism (category E) in the SC group at 35 and 42 days of age (P < 0.05).
Table 2
COG function of classes identified in feces of piglets at 16 days of age
COG_class
|
Description
|
CON
|
SC
|
P-value
|
D
|
Cell cycle control, cell division, chromosome partitioning
|
1.24 ± 0.10
|
1.38 ± 0.03
|
< 0.001***
|
O
|
Post-translational modification, protein turnover, and chaperones
|
2.96 ± 0.13
|
3.17 ± 0.17
|
< 0.001***
|
T
|
Signal transduction mechanisms
|
5.51 ± 0.34
|
4.78 ± 0.41
|
< 0.001***
|
V
|
Defense mechanisms
|
2.73 ± 0.18
|
2.86 ± 0.16
|
0.021*
|
A
|
RNA processing and modification
|
0.00 ± 0.01
|
0.00 ± 0.01
|
0.020*
|
J
|
Translation, ribosomal structure and biogenesis
|
6.19 ± 0.49
|
7.26 ± 0.22
|
< 0.001***
|
K
|
Transcription
|
8.51 ± 0.54
|
8.06 ± 0.53
|
0.012*
|
L
|
Replication, recombination and repair
|
6.44 ± 0.40
|
7.25 ± 0.18
|
< 0.001***
|
F
|
Nucleotide transport and metabolism
|
2.94 ± 0.19
|
3.23 ± 0.12
|
< 0.001***
|
G
|
Carbohydrate transport and metabolism
|
9.64 ± 0.66
|
8.32 ± 0.55
|
< 0.001***
|
H
|
Coenzyme transport and metabolism
|
4.05 ± 0.24
|
4.25 ± 0.32
|
0.032*
|
P
|
Inorganic ion transport and metabolism
|
4.81 ± 0.32
|
4.50 ± 0.18
|
0.001***
|
Q
|
Secondary metabolites biosynthesis, transport, and catabolism
|
1.10 ± 0.11
|
1.03 ± 0.08
|
0.013*
|
R
|
General function prediction only
|
11.64 ± 0.33
|
11.47 ± 0.15
|
0.048*
|
Note. The data are presented as means ± SD. CON, control group; SC, social contact. No superscript represents no significant difference (P > 0.05), *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively. |
Table 3
COG function of classes identified in feces of piglets at 35 days of age
COG_class
|
Description
|
CON
|
SC
|
P-value
|
M
|
Cell wall/membrane/envelope biogenesis
|
6.89 ± 0.54
|
6.31 ± 0.55
|
0.002**
|
O
|
Post-translational modification, protein turnover, and chaperones
|
3.14 ± 0.17
|
2.97 ± 0.12
|
0.001**
|
U
|
Intracellular trafficking, secretion, and vesicular transport
|
1.94 ± 0.16
|
1.67 ± 0.20
|
< 0.001***
|
V
|
Defense mechanisms
|
2.78 ± 0.13
|
2.99 ± 0.08
|
< 0.001***
|
W
|
Extracellular structures
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.011*
|
A
|
RNA processing and modification
|
0.00 ± 0.00
|
0.00 ± 0.00
|
< 0.001***
|
K
|
Transcription
|
8.23 ± 0.58
|
8.88 ± 0.41
|
< 0.001***
|
L
|
Replication, recombination and repair
|
7.05 ± 0.23
|
7.24 ± 0.19
|
0.008**
|
C
|
Energy production and conversion
|
5.50 ± 0.23
|
5.31 ± 0.17
|
0.007**
|
G
|
Carbohydrate transport and metabolism
|
8.36 ± 0.46
|
8.67 ± 0.25
|
0.014*
|
H
|
Coenzyme transport and metabolism
|
4.19 ± 0.25
|
4.00 ± 0.15
|
0.007**
|
I
|
Lipid transport and metabolism
|
2.45 ± 0.07
|
2.32 ± 0.09
|
< 0.001***
|
P
|
Inorganic ion transport and metabolism
|
4.58 ± 0.23
|
4.32 ± 0.09
|
< 0.001***
|
Q
|
Secondary metabolites biosynthesis, transport, and catabolism
|
1.08 ± 0.07
|
0.99 ± 0.05
|
< 0.001***
|
Note. The data are presented as means ± SD. CON, control group; SC, social contact group. No superscript represents no significant difference (P > 0.05), *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively. |
Table 4
COG function of classes identified in feces of piglets at 42 days of age
COG_class
|
Description
|
CON
|
SC
|
P-value
|
M
|
Cell wall/membrane/envelope biogenesis
|
6.41 ± 0.24
|
6.07 ± 0.22
|
< 0.001***
|
N
|
Cell motility
|
1.22 ± 0.27
|
0.95 ± 0.21
|
0.002**
|
O
|
Post-translational modification, protein turnover, and chaperones
|
3.04 ± 0.87
|
2.91 ± 0.06
|
< 0.001***
|
T
|
Signal transduction mechanisms
|
5.44 ± 0.30
|
5.20 ± 0.29
|
0.021 *
|
U
|
Intracellular trafficking, secretion, and vesicular transport
|
1.73 ± 0.10
|
1.56 ± 0.13
|
< 0.001***
|
V
|
Defense mechanisms
|
3.03 ± 0.07
|
2.95 ± 0.08
|
0.008**
|
W
|
Extracellular structures
|
0.00 ± 0.00
|
0.00 ± 0.00
|
0.023*
|
Z
|
Cytoskeleton
|
0.01 ± 0.00
|
0.01 ± 0.00
|
< 0.001***
|
B
|
Chromatin structure and dynamics
|
0.01 ± 0.00
|
0.02 ± 0.00
|
0.004**
|
L
|
Replication, recombination and repair
|
7.30 ± 0.20
|
7.56 ± 0.36
|
0.019*
|
E
|
Amino acid transport and metabolism
|
7.75 ± 0.16
|
7.99 ± 0.13
|
< 0.001***
|
F
|
Nucleotide transport and metabolism
|
3.09 ± 0.09
|
3.26 ± 0.20
|
0.006**
|
G
|
Carbohydrate transport and metabolism
|
8.78 ± 0.32
|
9.04 ± 0.25
|
0.011*
|
H
|
Coenzyme transport and metabolism
|
4.04 ± 0.08
|
3.84 ± 0.12
|
< 0.001***
|
I
|
Lipid transport and metabolism
|
2.36 ± 0.06
|
2.31 ± 0.05
|
0.014*
|
R
|
General function prediction only
|
11.38 ± 0.12
|
11.50 ± 0.08
|
0.003**
|
S
|
Function unknown
|
6.93 ± 0.10
|
7.37 ± 0.34
|
< 0.001***
|
Note. The data are presented as means ± SD. CON, control group; SC, social control group. No superscript represents no significant difference (P > 0.05), *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively. |
Table 5
COG function of classes identified in feces of piglets at 63 days of age
COG_class
|
Description
|
CON
|
SC
|
P-value
|
M
|
Cell wall/membrane/envelope biogenesis
|
6.61 ± 0.47
|
6.26 ± 0.18
|
0.005**
|
O
|
Post-translational modification, protein turnover, and chaperones
|
3.12 ± 0.15
|
2.98 ± 0.06
|
< 0.001***
|
U
|
Intracellular trafficking, secretion, and vesicular transport
|
1.73 ± 0.09
|
1.66 ± 0.10
|
0.033*
|
Z
|
Cytoskeleton
|
0.01 ± 0.00
|
0.01 ± 0.00
|
0.032*
|
B
|
Chromatin structure and dynamics
|
0.01 ± 0.00
|
0.02 ± 0.00
|
0.006**
|
K
|
Transcription
|
8.58 ± 0.45
|
8.83 ± 0.19
|
0.034*
|
C
|
Energy production and conversion
|
5.20 ± 0.13
|
5.07 ± 0.15
|
0.011*
|
E
|
Amino acid transport and metabolism
|
7.75 ± 0.24
|
7.88 ± 0.09
|
0.035*
|
G
|
Carbohydrate transport and metabolism
|
8.42 ± 0.30
|
8.83 ± 0.33
|
0.001**
|
H
|
Coenzyme transport and metabolism
|
4.12 ± 0.17
|
3.85 ± 0.13
|
< 0.001***
|
I
|
Lipid transport and metabolism
|
2.44 ± 0.11
|
2.37 ± 0.09
|
0.035*
|
P
|
Inorganic ion transport and metabolism
|
4.29 ± 0.14
|
4.15 ± 0.07
|
< 0.001***
|
S
|
Function unknown
|
6.98 ± 0.25
|
7.22 ± 0.26
|
0.007**
|
Note. The data are presented as means ± SD. CON, control group; SC, social contact group. No superscript represents no significant difference (P > 0.05), *, **, and *** represent P < 0.05, P < 0.01, and P < 0.001, respectively. |
To predict the probable functional changes induced by the social contact, next-generation sequencing reads were further analyzed to determine the third level of the KEGG pathways. As seen in Fig. 6, the social contact did not affect the microbial abundance of KEGG pathways at 16 days of age (P > 0.05). However, at 35, 42, and 63 days of age, piglets in the SC group had a lower enrichment of KEGG pathways involved in: Arachidonic acid metabolism, Citrate cycle (TCA cycle), Glycan biosynthesis and metabolism, Glycine, serine and threonine metabolism, Nicotinate and nicotinamide metabolism, Oxidative phosphorylation, Phenylalanine metabolism, Protein digestion and absorption, Riboflavin metabolism, Vitamin B6 metabolism, Tropane, piperidine, and pyridine alkaloid biosynthesis as well as Steroid hormone biosynthesis. While other pathways including: Galactose metabolism, Glycerolipid metabolism, Glycolysis/Gluconeogenesis, Pentose phosphate pathway, Pyruvate metabolism, Replication, recombination and repair proteins, Transcription factors, and Transporters were higher in the CON group at 35, 42, and 63 days of age.
Weight Of Piglets
As shown in Fig. 7, there was no significant difference in the weight at the age of 35 days between the CON and SC groups (P > 0.05), but a significant decrease was observed in the weight for the SC group compared to the CON group at 63 days of age (P < 0.05).