Rarefaction curves
In this experiment, 10 samples obtained were sequenced and analyzed. A total of 873 733 optimized sequences and 771 878 valid sequences were obtained, and a total of 2 280 OTUs were defined. The rarefaction curve began to flatten at around 10,000, indicating that the number of sequencing is gradually reasonable, and the increase in the number of sequencing has little contribution to the discovery of new OTUs. (Fig. 1) In addition, the Shannon Index is used to indicate the diversity of microorganisms in the sample. The curve of each sample tends to be flat, indicating that the sample covers most of the microbial information. Microbial diversity in the sample can be fully displayed, and the reliability of subsequent analysis can be guaranteed. (Fig. 2)
Alpha diversity analyses
The Alpha diversity index of the African ostrich colon is shown in Table 2, and the statistical histogram is shown in Fig. 3. Combining Table 2 and Fig. 3, the abundance indexes ACE and Chao1 both increased first and then decreased with increasing age, reaching the highest at 60 days of age (ACE 271.5, Chao1 274.097), and compared with other ages, 60 days old has the highest microbial richness. ACE and Chao1 indexes were the lowest at 180 days of age, 209.465, and 210, respectively, and the microbial abundance was the lowest. The diversity index Shannon index gradually decreases with age, while the Simpson index shows the opposite trend, indicating that the microbial diversity in the African ostrich colon decreases with age, and the diversity is highest at 7 days of age, the lowest diversity at 180 days of age.
Table 2
The colonic microbial diversity index of African ostriches at different ages
Sample ID | ACE | Chao1 | Shannon | Simpson |
E1 (7-day-old) | 241.790 ± 5.973 | 251.303 ± 6.831 | 3.203 ± 0.229 | 0.104 ± 0.034 |
E2 (30-day-old) | 259.887 ± 21.542 | 267.139 ± 22.808 | 3.173 ± 0.099 | 0.130 ± 0.016 |
E3 (60-day-old) | 271.500 ± 19.444 | 274.097 ± 21.476 | 2.768 ± 0.041 | 0.173 ± 0.009 |
E4 (180-day-old) | 209.465 | 210.000 | 1.388 | 0.374 |
The composition of the flora in the colon of African ostriches of different ages
Analyzed at the phylum level, there are differences in the composition and abundance of flora in the colon of African ostriches of different ages. When the African ostrich is 7 days old, there are differences in the composition and abundance of the flora in different intestinal segments of the colon. The phyla with higher abundance in the anterior and middle segment of the colon are Firmicutes, Tenericutes, Bacteroidetes, and Verrucomicrobia, and Firmicutes have the highest relative abundance, 52.8%, and 51.2%, respectively. The relative abundance of Proteobacteria in the front and middle segment is extremely low, 1.1% and 2.1%, respectively. In the 7-day-old posterior segment of the colon, Proteobacteria is the most important phylum, with a relative abundance of 39.7%, which is significantly higher than the anterior and middle segments. Firmicutes is the second dominant species, accounting for 37.8%, significantly lower than the anterior and middle segment. In addition, the relative abundance of Bacteroidetes and Verrucomicrobia in the posterior segment is also significantly lower than the relative abundance of the anterior and middle segments. At the age of 30 days, the composition and abundance of the intestinal segments of the colon are not much different, and they are mainly composed of Firmicutes, Tenericutes, and Bacteroidetes. In addition, each segment has a certain abundance of Proteobacteria, and the anterior segment is significantly higher than the middle and posterior segments. At the age of 60 and 180 days, the Proteobacteria was the most dominant phylum in the colon, with an average relative abundance of 51.7% and 56.5%, respectively, followed by Firmicutes, accounting for 25.9% and 42.9%. It can be seen that the abundance of Firmicutes at 180 days is significantly higher than that at 60 days. At 60 days of age, in the colon, besides the Proteus and Firmicutes, there are also the phylum Tenericutes, Bacteroidetes and Verrucomicrobia, which have high abundance, while in the colon at 180 days of age, the Proteobacteria and Firmicutes the relative abundance have exceeded 98%, and the abundance of other bacteria phyla are extremely low. (Fig. 4)
At the genus level, at 7 days of age, the colon is mainly composed of Anearoplasma, Bacteroides, Akkermania, Escherichia-Shigella, and in its posterior colon, the relative abundance of the Acinetobacter is 35.8%, which is significantly higher than the former and middle segment. The microorganisms in the colon at the age of 30 days are mainly classified into Anearoplasma, Bacteroides, Christensenellaceae-R-7-group, Acinetobacter, and Escherichia-Shigella, among which Anearoplasma is the most dominant genus in each segment, with an average relative abundance of 15.9%. There was no significant difference in the composition and relative abundance of the bacterial communities of each segment of the colon at the age of 60 days, mainly composed of Acinetobacter, Anearoplasma, Bacteroides, Christensenellaceae-R-7-group, and Akkermania. Acinetobacter is the most dominant genes, with an average relative abundance of 50.9%. At 180 days of age, the composition of the colon flora is concentrated in Pseudomonas, and its relative abundance reached 56.1%. (Fig. 5)
PCA analyses
Perform principal component analysis based on the OTU types and abundance obtained from all samples, and draw PCA diagrams. As showed in Fig. 6, except for the larger points of dispersion in the 7-day-old group, the dispersion in the 30-day and 60-day-old groups is smaller. It shows that the composition of the colon flora is quite different at the age of 7 days. The composition of the colonic colon at 30 days and 60 days is similar. Among the different ages, the 7-day and 30-day age graphs are closer, and both are farther away from the 60-day and 180-day age. It shows that the composition of colonic flora at 7-day and 30-day-old is relatively similar and is quite different from that of 60-day and 180-day-old. The degree of dispersion between 60-day-old and 180-day-old is relatively large, showing a large difference in floral composition.
LEfSe analyses of the differential flora in the colon contents of ostriches of different ages
The results showed that there were different flora in the colon of the African ostrich of different ages. At the age of 180 days, the Planococcaceae have a higher abundance. At 60 days of age, there are 36 bacterial groups, including Ruminococcaceae-UCG-011, Christensenellaceae_R-7_group, Christensenellaceae, Gordonibacter, Synergistia, Synergistetes, Aerococcus, Ruminococcaceae-NK4A214_group, Lachnoclostridium, Family-XIII, Family-XIII-AD3011-group, Exiguobacterium, Clostridium-sensu-stricto-6, Erysipelotrichaceae-UCG-004, Eubacterium-coprostanoligenes-group, etc. have a high abundance. At 30 days of age, there are 37 bacterial groups with high abundance, including Anaeroplasma, Anaeroplasmataceae, Tenericutes, Mollicutes, Clostridiales, Lachnospiraceae, Clostridiales-vadinBB60-group, Coprococcus-3, Ruminiclostridium-5, Ruminiclostridium-9, Lachnospiraceae-ND3007-group, Angelakisella, Acetitomaculum, Muribaculaceae, Lachnospiraceae-NK4A136-group, Lachnospiraceae-FCS020-group, Enterococcus, Cyanobacteria, etc. At 7 days of age, there are 34 bacterial groups with high abundance, including Erysipelotrichaceae, Lachnoclostridium-10, Mollicutes-RF39, Subdoligranulum, Lachnospiraceae-NC2004-group, Ruminococcaceae-UCG-013, T2WK15B57, Odoribacter, Marinifilaceae, Coprococcus-1, Ruminococcus-gauvreauii-group, Barnesiellaceae, Ruminococcus-2, etc. (Fig. 7)