Changes in the intestinal microbiota of Cobb broilers at different growth stages
The 16S rDNA amplicon sequencing was used to analyze the intestinal microbiota within the feces, jejuna, and ceca of Cobb broilers at different growth stages. After merging quality-filtered reads obtained from Illumina NovaSeq sequencing, a total of 2,254,549 effective sequence tags (median: 59,722 per sample; range of 36,084–69,463) were retained. (Fig. 1a-c) shows the longitudinal changes in intestinal microbial community diversity within Cobb broilers of different ages. The one-day-old Cobb broilers exhibited significantly lower fecal microbial diversity than those from other ages, reflecting an initial state of intestinal microbial community establishment. Feces exhibited high microbial diversity on day 14, but sharply decreased on days 28 and 35, followed by another increase on day 42. The jejunal communities exhibited relatively high microbial diversity on day 14, but decreased diversity on days 28, 35, and 42. Ceca communities exhibited similar microbial diversity levels on days 14 and 35 that were lower than those observed on days 28 and 42. The above results indicate longitudinal changes in microbial community diversity within the three intestinal components analyzed here.
PCoA analysis of Bray-Curtis distances (Fig. 1d-f) revealed significant differences in the intestinal microbial community structures of Cobb broilers at different growth stages. In particular, the fecal microbiota compositions of 42-day-old Cobb broilers were significantly different from those at other ages. The jejunal microbiota compositions were significantly different between communities in the 14–28- and 35–42-day-old broilers. Further, the cecal microbiota communities exhibited differences across the four growth stages. The 10 most abundant phyla and genera were used to investigate differences in the means of taxonomic relative abundances within the fecal, jejunal, and cecal microflora among the 5 different treatment groups (Fig. 2). At the phylum level, Firmicutes were dominant in the broiler intestinal microflora, followed by the Proteobacteria that accounted for relatively high proportions of fecal and jejunal communities, and the Bacteroidetes that accounted for relatively high proportions of cecal microflora. However, the abundances of the above taxa exhibited significant differences among different broiler growth stages. Firmicutes abundances in feces and ceca slowly increased from day 14 to 42. In addition, the Lactobacillus genus accounted for a relatively minor proportion of the feces of 1-day-old broilers, which significantly differed from their abundances at other time points. In contrast, Lactobacillus was the dominant genus in broiler feces and jejuna from 14 to 42 days, while they were completely dominant in ceca only at day 35. Further, Faecalibacterium, Alistipes, and Bacteroides accounted for higher proportions of cecal communities, although their abundances were significantly different among different broiler growth stages.
Distinct microflora members within broilers of different growth stages
An LDA score of 4.0 was used for LEfSe analysis at the phylum to species taxonomic levels in order to determine specific populations associated with broilers at different growth stages (Fig. 3). A total of 29 significantly different representative taxa were identified in the feces. Among them, 10 species, including Enterococcus faecium, Sphingobacterium mizutaii, and Lactobacillus amylotrophicus, were significantly enriched in 1-day-old broiler feces. Ten microbial taxa were also significantly enriched in 14-day-old Cobb feces, including Ralstonia pickettii, Klebsiella pneumoniae, and Lachnospiraceae. Only Lactobacillus johnsonii was significantly enriched in 28-day-old broiler feces. Lactobacillus salivarius was significantly enriched in 35-day-old broiler feces and significant enrichment of a single lineage in 42-day-old broiler feces was observed for a lineage associated with the Firmicutes, Bacilli, Lactobacillales, Lactobacillaceae, Lactobacillus, and Lactobacillus aviarius. A total of 12 significantly differential taxa were observed for the jejunal communities including the Enterobacterales and Lactobacillus reuteri that were significantly enriched in 14-day-old broiler jejunal samples. Significant enrichment was observed for 1 lineage of organisms in the 28-day-old broiler jejunal communities comprising Gammaproteobacteria, Burkholderiales, Burkholderiaceae, Ralstonia, and Ralstonia pickettii. In addition, Acinetobacter radioresistens abundances were significantly enriched in 35-day-old broiler jejunal communities. The taxa Lactobacillus aviaries, Alistipes onderdonkii, Barnesiella, and DTU089 were significantly enriched in 42-day-old broiler jejunum communities. In the cecal communities, 34 significantly differential representative taxa were identified. Among these, Bacteroidales and Clostridia were significantly enriched in 14-day-old broiler ceca. In addition, six microbial taxa, including Alistipes inops, Bacteroides fragilis, and Oscillospiraceae, were significantly enriched in 28-day-old broiler ceca. Further, 3 lineages were significantly enriched in 35-day-old broiler ceca, namely: (1) Bacilli, Lactobacillales, Lactobacillaceae, Lactobacillus, and Lactobacillus aviaries, (2) Anaerolineae and unidentified Anaerolineae, and (3) Barnesiella. Likewise, 2 lineages were significantly enriched in 42-day-old broiler ceca, namely: (1) Tannerellaceae, Parabacteroides, and Parabacteroides sp. CT06; and (2) Clostridia, Oscillospirales, Ruminococcaceae, and Faecalibacterium. The above results demonstrated that the intestinal microbial communities of Cobb broilers developed with age.
To further investigate differences of microbial communities among different intestinal sites, the three components (jejuna, ceca, and feces) of 42-day-old broilers were used for LEfSe analysis with an LDA threshold score of 4.0 (Fig. 4). There were relatively greater representative bacteria in feces, with 19 taxa (e.g., Bacteroidales, Clostridia, and Helicobacter) significantly enriched in those communities. The jejuna exhibited the next most numbers of representative taxa with three lineages that were significantly enriched including (1) Gammaproteobacteria, Enterobacterales, Enterobacteriaceae, Escherichia-Shigella, and Escherichia coli, (2) Lactobacillus johnsonii, and (3) Lactobacillus reuteri. Only one lineage of taxa was significantly enriched in the ceca: Bacilli, Lactobacillales, Lactobacillaceae, Lactobacillus, and Lactobacillus aviarius.
Regulatory effects of EOA1 and EOA2 on Cobb broiler intestinal microbiota
We previously observed that diets supplemented with EOA1 or EOA2 could improve broiler growth performance, with effects similar to the use of antibiotics Table S2. In this study, we investigated changes in intestinal microbial community diversity of Cobb broilers at different time points after supplementing diets with EOA1 and EOA2. A total of 10,474,315 effective high-quality sequence tags (36,084–69,576 per sample) were obtained for subsequent analysis. ɑ-diversity indices Table 1 of fecal, jejunal, and cecal communities were analyzed for each group of broilers at 14, 28, 35, and 42 days of age. The coverage index estimates for the broiler feces, jejunal, and cecal communities at different growth stages in each group were nearly 99%, indicating that the sequencing depth used here was adequate to detect native diversity within the samples. At 14 days of age, the fecal community Shannon index values were significantly lower in the EOA1 group than in the control and VM groups (p < 0.05). The cecum community Shannon index values were significantly higher in the EM group than in the other four groups (p < 0.05), while the cecum Shannon index values were significantly lower in the control group than in the other four groups (p < 0.05). At 28 days of age, the fecal community Shannon index values in the EM group were lower than in the EOA2 group (p < 0.05). In addition, the jejunal community Shannon index values of the control, VM, and EOA2 groups were significantly lower than in the EOA1 group (p < 0.05). Further, the cecum community Shannon index values in the EOA1 and EOA2 groups were significantly higher than in the other three groups (p < 0.05), while the cecum community Shannon index values in the VM group were lower than in the control and EM groups (p < 0.05). In broilers at 35 days of age, the fecal community Shannon index values were significantly higher in the EOA2 group than in the control group (p < 0.05). In contrast, the jejunal community Shannon index values were significantly lower in the control and VM groups compared to the EM and EOA1 groups (p < 0.05), while the cecum community Shannon index values were significantly higher in the EOA2 group than in the VM and EOA1 groups (p < 0.05). Considering the 42-day-old broilers, the lowest fecal community Shannon index value was observed for the EM group. These results indicate that diets supplemented with EOA1 or EOA2 can significantly alter the cecal, jejunal, and fecal microbial community diversity in Cobb broilers, while these effects are also influenced by broiler age.
Table 1
Effect of different treatment groups on the intestinal microbial alpha diversity index of Broilers1.
Items
|
Groups
|
Control
|
VM
|
EM
|
EOA1
|
EOA2
|
SEM2
|
P
|
Day 14
|
Feces
|
Shannon
|
4.50a
|
4.35a
|
4.05ab
|
2.79b
|
3.90ab
|
0.24
|
0.149
|
Goods coverage
|
0.9930b
|
0.9943ab
|
0.9933ab
|
0.9980a
|
0.9980a
|
0.0008
|
0.077
|
Jejunum
|
Shannon
|
2.01
|
1.67
|
2.16
|
2.24
|
2.21
|
0.18
|
0.888
|
Goods coverage
|
0.9983
|
0.9980
|
0.9977
|
0.9973
|
0.9983
|
0.0003
|
0.772
|
Cecum
|
Shannon
|
5.16c
|
6.01b
|
6.55a
|
5.73b
|
6.03b
|
0.13
|
0.001
|
Goods coverage
|
0.9980a
|
0.9980a
|
0.9933b
|
0.9980a
|
0.9980a
|
0.0006
|
0.019
|
Day 28
|
Feces
|
Shannon
|
2.74ab
|
2.78ab
|
2.25b
|
2.45ab
|
2.88a
|
0.09
|
0.110
|
Goods coverage
|
0.9977
|
0.9980
|
0.9983
|
0.9980
|
0.9980
|
0.0001
|
0.351
|
Jejunum
|
Shannon
|
2.19b
|
1.64c
|
2.35ab
|
2.50a
|
2.06b
|
0.09
|
0.001
|
Goods coverage
|
0.9990
|
0.9990
|
0.9987
|
0.9990
|
0.9993
|
0.0001
|
0.351
|
Cecum
|
Shannon
|
6.43b
|
5.75c
|
6.63b
|
7.26a
|
7.18a
|
0.16
|
0.000
|
Goods coverage
|
0.9980a
|
0.9980a
|
0.9947b
|
0.9933b
|
0.9920b
|
0.0008
|
0.004
|
Day 35
|
Feces
|
Shannon
|
2.27b
|
2.54ab
|
2.73ab
|
2.71ab
|
3.00a
|
0.09
|
0.050
|
Goods coverage
|
0.9980
|
0.9973
|
0.9980
|
0.9980
|
0.9983
|
0.0002
|
0.785
|
Jejunum
|
Shannon
|
1.70b
|
1.65b
|
2.49a
|
2.29a
|
2.04ab
|
0.11
|
0.013
|
Goods coverage
|
0.9980
|
0.9980
|
0.9987
|
0.9987
|
0.9990
|
0.0002
|
0.205
|
Cecum
|
Shannon
|
5.26abc
|
4.67c
|
5.66ab
|
5.07bc
|
5.92a
|
0.15
|
0.021
|
Goods coverage
|
0.9970a
|
0.9970a
|
0.9970a
|
0.9920ab
|
0.9873b
|
0.0013
|
0.024
|
Day 42
|
Feces
|
Shannon
|
3.30ab
|
3.98a
|
2.86b
|
3.69a
|
3.39ab
|
0.13
|
0.047
|
Goods coverage
|
0.9977a
|
0.9977a
|
0.9980a
|
0.9927b
|
0.9933b
|
0.0008
|
0.019
|
Jejunum
|
Shannon
|
1.02
|
1.34
|
1.20
|
1.47
|
1.56
|
0.08
|
0.247
|
Goods coverage
|
0.9990
|
0.9990
|
0.9990
|
0.9990
|
0.9990
|
0.0000
|
|
Cecum
|
Shannon
|
6.23
|
5.92
|
6.44
|
6.31
|
6.95
|
0.17
|
0.456
|
Goods coverage
|
0.9947
|
0.9973
|
0.9980
|
0.9973
|
0.9923
|
0.0009
|
0.284
|
1Value with different small letters mean significant difference (P < 0.05).
2SEM, standard error of means.
To identify specific intestinal taxa within broilers of different treatment groups, LEfSe analysis was used to compare communities among groups. At 14 days of age, 11 microbial taxa exhibited significantly different abundances in the feces of groups, with one microbial taxon significantly associated with the jejunum and 38 microbial taxa that were significantly associated with the ceca (Fig. 5a, 6a, 7a). After supplementing diets with VM, 14-day-old broilers exhibited significantly higher abundances of Enterococcus faecium, Aerococcus, and Aerococcaceae in feces, while Lactobacillus reuteri abundances in the jejuna were significantly lower. Significantly enriched flora in the ceca were associated with Clostridia (primarily including Lachnospiraceae, Oscillibacter, Butyricicoccaceae, Ruminococcaceter, and the [Eubacterium] coprostanoligenes group). Five microbial taxa were significantly enriched in the ceca of 14-day-old broilers in the EM group, including Faecalibacterium prausnitzii, Bifidobacterium, and Actinobacteriota. When EOA1 group broilers were 14 days old, Enterococcaceae, Enterococcus, and Enterococcus cecorum were significantly enriched in feces, and significantly enriched taxa in the ceca were associated with the Bacilli (including the Clostridia vadinBB60, UCG__005, and Ruminococcaceae groups). Lactobacillus aviarius and Lactobacillus johnsonii were significantly enriched in the feces of 14-day-old broilers in the EOA2 group, while 10 taxa were significantly enriched in the cecal communities including E. coli, Oscillospiraceae, and Faecalibacterium. Thirteen microbial taxa were significantly enriched in the feces of 28-day-old broilers, while 17 were significantly enriched in jejuna, and 32 were significantly enriched in ceca (Fig. 5b, 6c, 7b). In the control group, 4 taxa (e.g., Lactobacillus phage Sal3 and Lactobacillus salivarius) were significantly enriched in the feces of 28-day-old broilers, and 8 taxa (e.g., Lactobacillus reuteri, Ralstonia pickettii, Lactobacillus salivarius, Campylobacteria, and Alcaligenaceae) were significantly enriched in the jejuna, and 5 taxa (e.g., Oscillospirales and Alistipes) were enriched in the ceca.
After diet supplementation with VM, the abundances of fecal Gordonibacter were significantly higher in 28-day-old broilers, while Lactobacillus aviarius was significantly enriched in jejunal communities, and four lineages were significantly enriched in the ceca, including (1) Alistipes sp. CHKCI003, (2) Lachnospirales, Lachnospiraceae, (3) Bacteroidota, Bacteroidia, Bacteroidales, Bacteroidaceae, Bacteroides, Bacteroides dorei, and (4) Lactobacillales, Lactobacillaceae, Lactobacillus. After diet supplementation with EM, 28-day-old broilers exhibited significant enrichment of Clostridium sp. AUH-JLC140, Lactobacillus aviaries, and unidentified Oscillospiraceae in the feces. In addition, seven taxa (e.g., Lolium perenne and Dietzia maris) were significantly enriched in jejunal communities, while Bacteroidaceae, Bacteroides, and Bacteroides fragilis were significantly enriched in cecal communities. Tannerellaceae, Parabacteroides, Parabacteroides merdae, RF39, and Proteobacteria were more abundant in the ceca of 28-day-old broilers in the EOA1 group. When broilers in the EOA2 group were 28 days old, five microbial taxa (e.g., Enterococcus, Kurthia sp. 11kri321 and Corynebacteriaceae) were significantly enriched in feces, while only Lactobacillus johnsonii was significantly enriched in jejuna, and seven microbial taxa (e.g., Alistipes inops, Coprobacter, and Rikenella microfusus) were significantly enriched in ceca. At 35 days of age, 2 microbial taxa were significantly enriched in the fecal and jejunal communities, while 14 microbial taxa were significantly enriched in the ceca (Figure. 5c, 6b, 7c). Four microbial taxa (e.g., Lactobacillus aviarius, Barnesiella, and the Clostridia vadinBB60 group) were significantly enriched in the ceca of the 35-day-old control group broilers. When broilers with VM supplemented in the diet were 35 days old, Lactobacillus salivarius was significantly enriched in the feces and Lactobacillus aviarius was significantly enriched in jejuna. After diet supplementation with EM in 35-day-old broilers, 5 microbial taxa (e.g., Oscillospiraceae, Gammaproteobacteria, and Enterobacteriaceae) were significantly enriched in their ceca. Likewise, 35-day-old broilers with diets supplemented with EOA1 exhibited significant enrichment of Lactobacillus salivarius in jejuna, in addition to significant enrichment of Parabacterioides merdae and Bacterium ic1379 in their ceca. Moreover, 35-day-old broilers with EOA2 diet supplementation exhibited significant enrichment of Lactobacillus aviarius in feces, but significant enrichment of Butyricicoccaceae, Butyricicoccus, and Butyricicoccus pullicaecorum in the ceca. No significantly enriched species were observed in 42-day-old broiler feces, while 13 taxa were significantly enriched in the jejuna, and 29 were significantly enriched in the ceca (Fig. 6d, 7d). Four microbial taxa (e.g., Oscillospirales, Faecalibacterium, and Parabacteroides_sp_CT06) were significantly enriched in the ceca of 42-day-old control broilers. In 42-day-old broilers with diet supplementation of VM, Lactobacillus salivarius were significantly enriched in the jejuna, and 13 microbial taxa (e.g., Alistipes sp. CHKCI003, Alistipes onderdonkii, Parabacteroides merdae, Rikenella, and Barnesiella) were significantly enriched in the ceca. In 42-day-old broilers with diet supplementation of EM, six microbial taxa (e.g. bacterium ic1277 and unidentified chloroplasts) were significantly enriched in jejuna, while Alistipes inops, Bacteroides dorei, and the Clostridia vadinBB60 groups were significantly enriched in the ceca. Forty-two day old EOA1 group broilers exhibited significant enrichment of one lineage (Firmicutes, Bacilli, Lactobacillales, Lactobacillaceae, Lactobacillus, and Lactobacillus johnsonii) in the jejuna, while the [Ruminococcus] torques group, Gammaproteobacteria, and Proteobacteria were significantly enriched in their ceca. Six microbial taxa were significantly enriched in the 42-day-old EOA2 broiler ceca including Lactobacillus, Lactobacillus aviaries, and Pseudomonadales.
To understand the effects of EOA1 and EOA2 dietary supplements on intestinal microbial community function, functional annotation was performed for the fecal, jejunal, and cecal communities for 42-day-old broilers. The 35 most microbial abundant functions were evaluated using level 2 annotations (Fig. 8). Diets supplemented with VM led to lower inferred abundances of functions involved in the categories of xenobiotic biodegradation and metabolism, transcription, membrane transport, cellular community prokaryotes, and signal transduction. In contrast, several functional categories were enriched including glycan biosynthesis and metabolism, transport and catabolism, biosynthesis of other secondary metabolites, and other biological processes in the ceca. Diets supplemented with EM led to lesser effects on fecal and cecal biological processes, but enhanced biological processes in the jejuna related to metabolism (e.g., metabolism of terpenoids and polyketides, energy metabolism, and metabolism of cofactors and vitamins). In contrast, these treatments were associated with decreased inferred biological process functions related to cellular processes, genetic information processing, and environmental information processing. Diets supplemented with EOA1 or EOA2 resulted in less enrichment of jejunal and cecal biological process functions, but increased abundances of functions involved in fecal lipid metabolism, enzyme families, and xenobiotics biodegradation and metabolism, but decreased enrichment of functions involved in nucleotide metabolism and replication and repair. Thus, EOA1 and EOA2 treatment generally enhanced certain biological pathways related to metabolism, but led to decreased abundances of inferred functions related to cellular processes, genetic information processing, and environmental information processing.