Analysis of Colon Microbial Diversity of African Ostriches at Different Ages

Background:The colon is the unique digestive organ of the African ostrich. It has a large microbial population, which plays an important role in the digestive process of the African ostriches. Methods: In order to understand the diversity of colon microbes in African ostriches, this study used metagenomics sequencing technology to sequence and analyze the colonic microbes of African ostriches at the age of 7, 30, 60 and 180 days. Results: The results showed that with the increase of age, the microbial richness index rst increased and then decreased, the highest at 60 days of age (ACE 271.5, Chao1 274.097), while the microbial diversity gradually reduced with the increase of age, the highest at 7 days ( Shannon 3.203, Simpson 0.104). At the phylum level, the dominant bacteria phylum at 7 days of age is Firmicutes, while at 30 days of age, the phyla Firmicutes and Tenericutes are dominant phyla, and the abundance of Proteobacteria is highest at 60 days of age and 180 days of age. At the genus level, the dominant bacteria in the colon at 7 and 30 days are Anaeroplasma and Bacteroides. Acinetobacter is the dominant genus at 60 days, and the dominant genus at 180 days is Pseudomonas. PCA analysis showed that the microbial composition in the 180-day-old colon was very different from other days. The microbial composition in the anterior and middle part of the colon at 7-day-old was similar to that of 30-day-old, while the microbial composition in the latter part was similar to that of 60-day-old. Results of LEfSe analysis showed that there were 34, 37, and 36 differential ora in the colon at 7, 30, and 60 days, respectively, while there was only one differential ora at 180 days of age. Conclusion: The results of the study showed that the composition of the microbial community in the colon of African ostriches of different ages is different and there are different ora, but the microbes in the colon are still mainly concentrated in the phylum Firmicutes, Tenericutes, and Proteobacteria,

colonic microorganisms. Lu et al. found that Firmicutes is the dominant phylum in piglet colon [6]. Guo et al. found that the colonic microbial diversity of breeding Jinfen white pigs was signi cantly higher than that of local Mashen pigs [7]. Hu et al. found that the microbiota in the colon has a certain connection with the occurrence of cardiovascular disease [8]. Ji et al. found that short-term feeding of high-nutrient diets can change the microbial content in the colon of some Huan Jiang Xiang pigs [9].
The African ostrich is the largest herbivorous bird in the world. Unlike other poultry, it has a well-developed colon, which is about 2.5 times the length of the small intestine, accounting for 84.2% of the large intestine [10], and its colon is the site with the highest degree of microbial accumulation. At present, research on colonic microorganisms has mainly focused on mammals such as pigs and mice. There is no report about the related research of African ostrich. Understanding the composition and succession of the colonic microorganisms of the African ostrich is of great signi cance to promote the growth and development of the African ostrich and improve its production performance. Therefore, this experiment uses metagenomics methods to sequence and analyze the diversity of microorganisms in the colon of African ostriches of different ages.

Sample Collection
Test animals selected 4 healthy African ostriches, which were 7 days, 30 days, 60 days, and 180 days.
Adjust the diet ratio according to the dietary structure of experimental animals of different ages and meet the requirements of the National Research Council (1994). Use 20% Urethane (1 g/kg) for anesthesia.
After general anesthesia, open the abdominal cavity and quickly remove the colon. Extract the contents from the front, middle and back positions of the colon into a 2 mL EP tube, collect 10 samples in total, and label each sample. The speci c group numbers are shown in Table 1. Entrust Annoroad Gene Technology (Beijing) Co., Ltd. to conduct microbial mutagenesis sequencing and biological information analysis of the resulting sequence, and the speci c process is as follows: Test procedure After extracting the total DNA of the sample, primers are designed according to the conserved regions, and sequencing adapters are added to the ends of the primers to perform PCR ampli cation, and the products are puri ed, quanti ed, and homogenized to form a sequencing library. The constructed library is rst subjected to library quality inspection, and the quali ed library is sequenced with Illumina HiSeq 2500.

Information analysis process
According to the overlap relationship between PE reads, the paired-end sequence data obtained by Hiseq sequencing is merged into a sequence Tag. At the same time, the quality of Reads and the effect of Merge is ltered to obtain Effective Tags.

Drawing Rarefaction curve
The rarefaction curve is to randomly select a certain number of sequences from a sample, count the number of species represented by these sequences, and construct a curve based on the number of sequences and species to verify whether the amount of sequencing data is su cient to re ect the sample species diversity. The rarefaction curve re ects the rate of emergence of new OTUs (new species) under continuous sampling: within a certain range, as the number of sequencing items increases, if the curve shows a sharp rise, it means that a large number of species have been discovered in the community; The curve tends to be at, indicating that the species in this environment will not increase signi cantly with the increase in the number of sequencing.
The Shannon diversity index curve is drawn using Mothur software and R language tools according to the Shannon index of each sample at different sequencing depths. The larger the Shannon index, the more OTU species and the richer the species, indicating that most of the microbial species information is covered in the sample. When the curve tends to be at, it indicates that the amount of sequencing data is large enough, and the OTU types will not increase with the increase in the amount of sequencing; if the curve does not tend to be at, it indicates that it is not saturated, and increasing the amount of data can reveal more OTU.

Alpha diversity analysis
Alpha diversity can re ect the species diversity within a single sample, and it has multiple metrics. Chao1 and Ace indexes are used to measure the abundance of the community. The Shannon index can re ect the diversity of the community, and the Simpson re ects the concentration of dominant populations in the community. Therefore, the larger the Chao1, Ace, and Shannon index values, and the smaller the Simpson, the higher the species diversity of the sample.

Analysis of species composition and abundance
Comparing the representative sequence of OTU with the microbial reference database, the species classi cation information corresponding to each OTU can be obtained, and then the composition of each sample community at each level (phyla, family, class, family, genus, species) is counted. First, use QIIME software to generate species abundance tables at different taxonomic levels, and then use R language tools to draw community structure diagrams at each taxonomic level of the sample.

Principal component analysis
Principal component analysis (PCA) is a technique used to analyze and simplify data sets. By decomposing the composition of different sample OTUs, the differences between multiple sets of data will be re ected on the two-dimensional coordinate graph, and the coordinate axis can best re ect the two characteristic values of the variance. The closer the distance between the two samples, the more similar the composition of the two samples.

LEfSe analysis of samples between groups
Line discriminant analysis (LDA) is used to nd statistically different ora in the colon of African ostriches of different ages. Use the LDA value distribution histogram to display species with LDA scores greater than the set value (the default setting is 2.0). The length of the histogram indicates the in uence of different species (LDA score). The longer the length of the histogram, the greater the in uence of species on the differences between groups.

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 de ned. The rarefaction curve began to atten 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 at, 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   The composition of the ora in the colon of African ostriches of different ages Analyzed at the phylum level, there are differences in the composition and abundance of ora 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 ora 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 signi cantly higher than the anterior and middle segments. Firmicutes is the second dominant species, accounting for 37.8%, signi cantly lower than the anterior and middle segment. In addition, the relative abundance of Bacteroidetes and Verrucomicrobia in the posterior segment is also signi cantly 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 signi cantly 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 signi cantly 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 signi cantly higher than the former and middle segment. The microorganisms in the colon at the age of 30 days are mainly classi ed 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 signi cant 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 ora 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 7day-old group, the dispersion in the 30-day and 60-day-old groups is smaller. It shows that the composition of the colon ora 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 ora 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 oral composition.
LEfSe analyses of the differential ora in the colon contents of ostriches of different ages

Discussion
The colonization and development of the microbial community in the animal intestines are not static but dynamic, and a diversi ed microecosystem can be formed in the rst few weeks after the animal is born [11]. Studies have shown that the composition of the intestinal microbiota of young animals is highly uctuating. But as age increases, they gradually stabilize in the end. In this process, the intestinal tract at different developmental stages will form a dominant ora that adapts to the developmental needs of this stage and plays a corresponding role [12]. The taxonomic annotation results of this study show that Firmicutes, Tenericutes, and Bacteroidetes are the dominant phyla in the colon of African ostriches at the age of 7 and 30 days. Proteobacteria and Firmicutes in the colon were the dominant phyla at 60 and 80 days of age. Studies have shown that Bacteroidetes are directly related to the body's protein metabolism and lipid metabolism [13]. The high abundance of Bacteroidetes may be adapted to the digestion of highprotein and high-nutrient diets of African ostriches at the juvenile stage. Verrucomicrobia with higher abundance appeared at 7 and 60 days of ages. Studies have shown that the increase in the abundance of Verrucobacterium may be related to the lack of peptidoglycan in the body [14]. Peptidoglycan has a variety of biological effects such as anti-infection, anti-tumor, and immune regulation. The lack of peptidoglycan in the body will lead to reducing immune function and cause the invasion of pathogenic bacteria [15]. This suggests that the changes in the microbial community in the intestine can re ect the health of the body at this time and the hidden risk of pathogenicity. In addition, at the genus level, Anaeroplasma is the dominant genus at 7 and 30 days old. Acinetobacter is the dominant genus at 60 days of age, and Pseudomonas is the dominant genus at 180 days of age. The results of LEfSe analysis show that there are different bacterial groups in the colon of each age, indicating that the abundance and composition of microorganisms in the intestine will change signi cantly at different stages of growth and development. This is consistent with the research of Yatsunenko et al. [12].
The results of the Alpha diversity analysis of this study showed that the abundance of microorganisms in the colon rst increased and then decreased with age, while the diversity gradually decreased with age. It showed the highest microbial diversity at 7 days of age and the lowest at 180 days of age. PCA analysis shows that the 180-day-old colonic microbial composition is very different from other days. The microbiological composition of the front part of the 7-day-old colon is similar to that of the 30-day-old colon, and the latter part is similar to the 60-day-old colon. This shows that the colonic microbial population of African ostriches has stabilized in the adult stage, and with age, the gut microbes are also changing.

Conclusion
The results of the study showed that the composition of the microbial community in the colon of African ostriches of different ages is different and there are different ora, but the microbes in the colon are still mainly concentrated in the phylum Firmicutes, Tenericutes, and Proteobacteria, which gives the intestinal tract and function of the African ostrich further research provides a theoretical basis.    Histogram of phylum horizontal species distribution Note: The histogram only shows the top ten species in abundance level, and other species are merged into Others and shown in the gure. E11: 7-day-old anterior colon; E12: 7-day-old middle colon; E13: 7-day-old posterior colon; E21: 30-day-old anterior colon; E22: 30 Day-old middle colon; E23: 30-day-old posterior colon; E31: 60-day-old anterior colon; E32: 60-dayold middle colon; E33: 60-day-old posterior colon ; E41: 180-day-old anterior colon Histogram of phylum horizontal species distribution Note: The histogram only shows the top ten species in abundance level, and other species are merged into Others and shown in the gure. E11: 7-day-old anterior colon; E12: 7-day-old middle colon; E13: 7-day-old posterior colon; E21: 30-day-old anterior colon; E22: 30 Day-old middle colon; E23: 30-day-old posterior colon; E31: 60-day-old anterior colon; E32: 60-dayold middle colon; E33: 60-day-old posterior colon ; E41: 180-day-old anterior colon Histogram of genus horizontal species distribution Note: The histogram only shows the top ten species in abundance level, and other species are merged into Others and shown in the gure. E11: 7-day-old anterior colon; E12: 7-day-old middle colon; E13: 7-day-old posterior colon; E21: 30-day-old anterior colon; E22: 30 Day-old middle colon; E23: 30-day-old posterior colon; E31: 60-day-old anterior colon; E32: 60-dayold middle colon; E33: 60-day-old posterior colon ; E41: 180-day-old anterior colon Histogram of genus horizontal species distribution Note: The histogram only shows the top ten species in abundance level, and other species are merged into Others and shown in the gure. E11: 7-day-old anterior colon; E12: 7-day-old middle colon; E13: 7-day-old posterior colon; E21: 30-day-old anterior colon; E22: 30 Day-old middle colon; E23: 30-day-old posterior colon; E31: 60-day-old anterior colon; E32: 60-dayold middle colon; E33: 60-day-old posterior colon ; E41: 180-day-old anterior colon