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Comparison of the gut microbiota composition between the wild and captive Tibetan wild ass (Equus kiang)

Abstract Aims The gut microbiota has a great effect on the health and nutrition of the host. Manipulation of the intestinal microbiota may improve animal health and growth performance. The objectives of our study were to characterize the faecal microbiota between wild and captive Tibetan wild asses and discuss the differences and their reasons. Methods and Results Through high‐throughput sequencing of the 16S rRNA V4‐V5 region, we studied the gut microbiota composition and structure of Tibetan wild asses in winter, and analysed the differences between wild and captive groups. The results showed that the most common bacterial phylum in Tibetan wild ass faeces samples was Bacteroidetes, while the phylum Firmicutes was dominant in captive Tibetan wild ass faecal samples. The relative abundance of Firmicutes, Tenericutes and Spirochaetes were significantly higher (P < 0·01) than in the wild groups. Conclusions Captivity reduces intestinal microbial diversity, evenness and operational taxonomic unit number due to the consumption of industrial food, therefore, increasing the risk of disease prevalence and affecting the health of wildlife. Significance and Impact of the Study We studied the effect of the captive environment on intestinal micro‐organisms. This article provides a theoretical basis for the ex‐situ conservation of wild animals in the future.


Introduction
The Qinghai-Tibetan plateau provides one of the most extreme environments for the survival of humans and other mammals . The Tibetan wild ass (Equus kiang) is a unique species on the Qinghai-Tibetan plateau and is widely distributed in Qinghai, Gansu, Xinjiang, Sichuan and Tibet (Wu and Yi 2000;Moehlman 2002). It is a key protected species in China and is listed in the International Union for Conservation of Nature Red List 2012 of threatened species. Intensive research has been performed regarding the conservation of this species (Joseph and Bard-Jorgen 2005;Yifan and Jianping 2006;Yin et al. 2007;St-Louis and Côt e 2009;Kefena et al. 2012;Dong et al. 2015;Guo et al. 2018). With the development of wildlife protection plans, the change in environment during ex-situ conservation comes with a change in animal health.
The microbial community of the gastrointestinal tract remains balanced in terms of species, quantity and location in healthy organisms. Animal intestines have large, diverse and dynamically changing bacterial communities that play important roles in host immunity, nutrient metabolism and energy acquisition (Yun et al. 2017). The composition of the mammalian gut microflora is associated with many environmental factors, among which living conditions are a major part (Guan et al. 2016).
Captive environments affect the composition of gut microbe in wild animals (Xenoulis et al. 2010;Guan et al. 2016Guan et al. , 2017. Changes in the intestinal microbe composition are associated with host health and disease (Quigley 2010;Costa et al. 2012;Morgan et al. 2012;Qin et al. 2012). Diet is a key factor affecting microbial diversity in the host gut (Ley et al. 2008;Yin et al. 2017;Qin et al. 2018). As industrial food consumption increases in humans and wildlife, each dietary change is accompanied by an adjustment of intestinal microbes, resulting in the loss or extinction of certain intestinal microbes . Recent studies have shown that diet-induced loss of microbial diversity can be amplified over generations, resulting in reduced intestinal microbial diversity and increased risk of population extinction (Sonnenburg et al. 2016).
Therefore, the objectives of our study were (i) to characterize the faecal microbiota between wild and captive Tibetan wild asses; (ii) to analyse the differences between faecal samples from different environments; (iii) discuss the causes for the differences, and finally, (iv) to explore the relationship between diet, gut flora and host health. The study of intestinal microbial diversity, which can be used to assess host health and related diseases, provide a theoretical basis for the future breeding or release of wild animals.

Materials and methods
Faecal samples from Tibetan wild asses living in the wild were collected from different regions of the headwaters of the Yellow River, Maduo County on Qinghai-Tibet Plateau in January 2018. A total of 140 wild Tibetan wild ass faecal samples were collected. All samples were collected after natural defecation. Animals were not scared, nor driven, and drugs were not used to promote defecation. Captive Tibetan wild ass faecal samples were collected from the Qinghai-Tibet Plateau wild animal park in January 2018. In total 28 captive Tibetan wild ass samples were collected. None of the animals had received anti-inflammatory drugs or antimicrobials within the last 3 months.
All sample collection processes were performed in accordance with the requirements of the authorizing ethics committee.
Genomic DNA from the samples were extracted by the CTAB method. DNA purity and concentration were monitored on a 1% agarose gel. DNA samples were diluted to 1 ng ll À1 using sterile water. Universal 16S PCR primers (515F, 5 0 -GTGCCAGCMGCCGCGGTAA-3 0 and 907R, 5 0 -CCGTCAATTCCTTTGAGTTT-3 0 ) were used to amplify the V4 and V5 regions of the 16S rRNA. All PCR reactions were carried out with Phusion â High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA). The polymerase chain reaction was carried out using the following mixture in a final volume of 30 ll: 10 ll of template DNA, 3 ll of each primer (6 lmol l À1 ), 15 ll of Phusion Master Mix (29) and 2 ll of ddH 2 O. Next, DNA was amplified using the following conditions: denaturation for 1 min at 98°C, followed by 30 cycles of 10 s at 98°C for denaturation, 30 s at 50°C for annealing and 30 s at 72°C for extension, as well as a final extension step at 72°C for 5 min.  of PCR products was estimated using 2% agarose gel electrophoresis. PCR products were then purified with the GeneJETTM Gel Extraction Kit (Thermo Scientific, Waltham, MA). The library was sequenced on an Ion S5 TM XL platform and 400 bp single-end reads were generated. The singleend method was used to construct a small fragment library for single-end sequencing. By cutting and filtering reads, OTUs (operational taxonomic units) were clustered and species annotation and abundance analysis were performed to reveal sample species composition.
Novogene was commissioned to complete all experiments (DNA extraction, PCR amplification, library preparation and sequencing) and data analysis.
All diversity indices in our samples were calculated with QIIME (ver. 1.9.1) and displayed with R software (ver. 2.15.3). In R, NMDS analysis was displayed using the vegan package, principal coordinates analysis (PCoA) was displayed using the WGCNA package, stat package and ggplot2 package. Cluster analysis was preceded by principal component analysis (PCA), which was applied to reduce the dimensionality of the original variables using the factor Mine R package and ggplot2 package. Crossgroup and intra-group differences were tested using the MRPP function in the vegan package.

Results
Eighty-one faecal samples from wild and captive Tibetan wild asses were selected for sequencing, of which 60 samples were from wild animals (DY, DC and DZ), classified as the wild group (DYW), and 21 samples were from captive animals (DD1, DD2, DD3), classified as the captive group (DDD). A total of 4 809 901 high-quality reads were obtained from wild group and classified into 3542 OTUs, while 1 693 293 high-quality reads were obtained from the captive group and classified into 3155 OTUs. The number of OTUs present in both the wild and captive groups was 2928, with 614 unique OTUs in the wild group, and 227 unique OTUs in the captive group.
The rarefaction curves and rank abundance curves of the wild and the captive Tibetan wild ass faecal samples ( Fig. 1) show the richness and evenness of the species in the samples. As the sample size increased, the number of observed species gradually stabilized and there were no further significant growth or fluctuations. The results show that the curve had reached a plateau and the sequencing data were reasonable. The number of samples in this study was sufficient to study the intestinal microbial diversity of Tibetan wild asses in the field and in captivity.
We detected a total of 27 phyla, 47 classes, 81 orders, 134 families and 241 genera from 81 Tibetan wild ass faecal samples. In the wild group, we detected 26 phyla, 44 classes, 74 orders, 117 families and 199 genera, while in the captive group, 26 phyla, 43 classes, 71 orders, 121 families and 204 genera were detected.
In the wild group, Bacteroidetes (42Á59%) was the predominant phylum, and Anaerovorax (2Á29%) was the predominant genus. In the captive group, Firmicutes (49Á74%) was the predominant phylum, and Streptococcus (4Á39%) was the predominant genus. In order to show the relative abundance of bacterial communities more intuitively, we have chosen the top 10 species for each sample or group and generated a percentage stacked histogram of relative abundance at the phylum and genus levels in  The alpha diversity indices (including Shannon, Simpson, Chao1, ACE, Goods_coverage) are shown in Table 1 (cut-off = 62 431). The Goods coverage index was above 99%, indicating a high level of diversity was found in the samples. The Shannon, Chao1 and ACE indices in the wild group were higher than in the captive group (P Shannon = 0Á01627 < 0Á05, PChao1 = 0Á000381 < 0Á01, P ACE = 0Á000838 < 0Á01), but the Goods coverage index in the wild group was significantly lower than that in the captive group (P = 0Á009368 < 0Á01).
The PCA plot (Fig. 3a) and the PCoA plots (Fig. 3b) showed that the wild and the captive group formed two distinct areas on the graph. The similarity of the community structure was higher and the composition was more similar. The similarity between the two groups was obviously smaller than within the samples. In the PCA plot, the wild the captive groups were obviously separated, meaning that the similarity between the groups was small.
We also used a nonmetric multidimensional scaling (NMDS) plot to analyse discrepancies between the groups. Weighted and nonweighted methods were used for NMDS analysis, resulting in stress values of 0Á088 and 0Á102, respectively, which are both <0Á2 indicating that NMDS can accurately differentiate the samples. NMDS is a nonlinear model, whether it is weighted analysis or nonweighted analysis, and the wild and captive groups were clearly separated. For individuals, different groups of individuals will also be clustered into the corresponding group, indicating that the difference between the two groups was quite remarkable (Fig. 3c). MRPP testing between the wild and captive groups was A = 0Á1136 > 0. The difference between the groups was greater than the difference within the groups, indicating that the study groups were reasonable. The significance of 0Á001 < 0Á01, showed that the wild group and the captive group were significantly different.
The Metastat method was used to test the microbial species abundance data for wild and captive faecal samples. According to the q value at the phylum level and genus there was a significant difference between the species (P < 0Á01), and a plot of the difference between the species can be seen in the abundance distribution box map (Figs 4 and 5).

Discussion
In the analysis of alpha diversity, the Shannon, Chao1 and ACE indexes of the wild group were larger than those of the captive group, which suggests that the bacterial diversity of gut microbes in the wild Tibetan wild ass population is significantly higher than for those individuals in captivity. Although the intestinal microbial diversity of the wild Tibetan wild ass was higher, fewer microbes were identified, and the exploration of wild animal intestinal flora has a broader prospect. The Bacteroides and Firmicutes phyla made up more than 80% of the total bacterial content. This is consistent with previous studies of intestinal microbial diversity in mammals (Eckburg et al. 2005;Mariat et al. 2009;Middelbos et al. 2010;Qin et al. 2010;Van den Abbeele et al. 2010;Zhu et al. 2011;Guan et al. 2016) and these organisms facilitate the digestion of cellulose and hemicellulose in food . However, the numbers of bacteria from these two phyla were significantly different in the different host groups (P < 0Á01). Bacteroidetes was the dominant phylum in the wild group, while Firmicutes was the dominant phylum in the captive group.
In winter, captive Tibetan wild asses are fed semi-dry oat grass (fiber content 353Á1 g kg À1 ), feed (protein 17Á5%, fat 2%) and carrots (proportional to 8 : 2 : 1), and more fat and protein may reduce microbial diversity and lead to an increase in the number of Firmicutes and Actinobacteria He et al. 2013;Cani 2018). Thus the diversity of the gut microbiota was significantly lower in the captive group than in the wild group, with higher numbers of Actinobacteria and Firmicutes (Middelbos et al. 2010), and lower numbers of Bacteroidetes. The wild Tibetan wild asses feed mostly on Gramineae, Leguminosae and Cyperaceae plants, including pedicularis, Stipa purpurea, Brylkinia caudate, Poa annua, Carex myosuroides and Potentilla chinensis (Yin et al. 2007;Dong et al. 2015). In the wild, due to food shortage, protein and fat intake decreased, and the Bacteroidetes content increased to help host to increase their nutrition. A disruption of the symbiosis between the microbiota and host is known as dysbiosis and is described in multiple chronic diseases, such as obesity and malnutrition (Castaner et al. 2018;Zhang et al. 2018;Jeong et al. 2019), neurological disorders (Kurokawa et al. 2018;Quagliariello et al. 2018;Sun and Shen 2018), inflammatory bowel disease (IBD) (Costa et al. 2012;Roche-Lima et al. 2018), metabolic syndrome (Zhao et al. 2018), cancer and other diseases (Katsimichas et al. 2018;Lu et al. 2018;Panebianco et al. 2018;Pulikkan et al. 2018;Zitvogel et al. 2018). We presume that the health of the wild group of Tibetan wild asses was better than the captive group. On the one hand, in the case of captivity, the feeding density is high and there is long-term contact with human beings, with a higher probability of zoonosis among animals in captivity, and generally poorer health than animals in the wild. On the other hand, the intestinal microbial composition and content of the captive group was greatly altered, which can present as qualitative changes, such as increased proportions of harmful bacteria and reduced levels of beneficial bacteria. The captive Tibetan wild asses had more Spirochaetes, Proteobacteria and Campylobacter; groups of bacteria that contain pathogens (Ludwig et al. 2010), Proteobacteria is closely related to IBD and Clostridium difficile infection. Campylobacter is the most frequent cause of foodborne disease. At same time, the captive group samples had a lower content of Bacteroidetes, the basal microbiota, which is one of the richest phyla in a healthy human body and its levels can be a predictor of an animal's health.
In summary, there were significant differences in gut microbial composition and structure between wild and captive Tibetan wild asses. We believe that food, bacterial content and animal health are connected and changes in the numbers of different bacteria play an important role for the host.
With the intake of large amounts of industrial food, the intestinal microbial diversity of captive Tibetan wild asses decreased, increasing the risk of disease. Other methods of  feeding that better approximate nature should be chosen to protect rare and endangered wildlife in a captive environment. The gut microbiota of the Tibetan wild ass is complex and this study of its composition and function is of great significance to the protection of the Tibetan wild ass. In addition, it is important to conduct more research to understand how environmental differences directly affect the diversity of bacteria in stool samples.