Through fecal analysis, we studied the gut microbiome structure and the metabolomic profiles in both wild (WCM) and captive Chinese monals (CCM). The results of this research indicated distinct differences of the fecal bacterial communities between the two groups, furthermore, we identified significant correlations between the fecal microbiota and metabolites.
The levels of microbiome alpha and beta-diversity from WCM were significantly higher than CCM, indicating a more diverse microbial community in the wild population. These differences may be the result of differences in diet composition, geographical ranges, energy utilization, climate conditions, and stress exposure in the WCM vs. the CCM [9, 11].
Proteobacteria was the dominant phylum in the feces of WCM followed by Firmicutes, while Firmicutes was relatively more abundant than Proteobacteria in the feces of the captive group. The characterization of gut bacteria in several studies reveals a dominance of Firmicutes and Bacteroidetes, a feature which appears to be typical for birds. The dominance of these phyla has been described in the ceca of turkeys (Meleagris gallopavo) , the feces of penguins (Spheniscidae)  and Japanese quail (Coturnix japonica) , as well as in the crop of hoatzin (Opisthocomus hoazin) . However, one study reports that Proteobacteria and Firmicutes are the dominant phyla in the feces of the Lady Amherst's pheasant (Chrysolophus amherstiae), Reeves's pheasant (Syrmaticus reevesii), and Cabot's tragopan (Tragopan caboti) , which is more consistent with our observations of the microbiome composition in the Chinese monal. Moreover, Bacteroidetes just accounted for 3.8% and 1.6%, respectively, in the WCM and CCM unlike what was observed in the turkey, penguin, quail, and hoatzin studies. Based on the results of this study, it would appear that the gut microbial composition of the Chinese monal is most reflective of that which has been detected in other species of the family Phasianidae; however, in the aforementioned cited research only two samples per group were collected and study details are lacking which highlights the need for further research on this subject.
A large diversity of gut microbes has been described as adaptive and beneficial, with diet being considered one of the most critical factors shaping gut microbial structures. The Chinese monal is an omnivorous bird. For wild populations, food is scarce in the winter months, due to their natural environment being at a high altitude and low temperatures. Whereas, the small captive population of monals receives a relatively steady diet with no change in environment except for exposure to outdoor seasonal climate conditions. Our results indicated a high abundance of Proteobacteria in WCM. Proteobacteria have greatly variable morphology and versatile functions , previous studies have demonstrated that increased an richness of Proteobacteria in the gut microbial flora is mainly related to energy accumulation [28–30], and is more abundant when the host animal has prolonged exposure to cold climates . We suspect our findings in WCM may be in response to their comparatively complex dietary composition, as well as to help them with cold weather and scarce food to get more energy.
In contrast to WCM, Firmicutes was identified as the dominant bacteria phylum in CCM. Compared with the WCM, we suspect captive individuals are fed a diet with a comparatively higher lipid content. Diet composition, as indicated in Table 1 showed a 6.2% crude fat in the commercial pelleted feed, which provides the base of the diet, in addition to corn which contains 3.6–5.3% crude fat (The data provided at the China Feed-database Information Network Centre: http://www.chinafeeddata.org.cn/picture/pdf/CFIC2019_1.pdf). In a 2014 study, mice were fed a high-fat diet, and the Firmicutes: Bacteroidetes ratio in mice gut microbiome increased after three weeks, with the abundance of Firmicutes increased and Bacteroidetes depleted . This finding is in accordance with many former studies, suggesting that a high-fat content in diet is one of the primary factors responsible for changes observed in gut microbe composition [7, 32]. Thus, the increased abundances of Firmicutes and the Firmicutes: Bacteroidetes ratio in CCW group (CCM 33.45 versus WCM 6.25) is likely the result of a higher lipid content in the diet.
Concerning the metabolome, we detected 58 significant metabolites that associated with 20 metabolic pathways. We found that a significantly higher fatty-acid content in feces of the CCM was related to unsaturated fatty acid and fatty acid biosynthesis. A previous study found that different dietary triacylglyceride composition has an influence on fatty acid content in the feces of human neonates. Thus, our finding may be related to the nutrient composition of the diet, which contains a higher level of fat and a different lipid composition than what wild specimens are likely consuming. Otherwise, our results showed that the primary microbial genera in Chinese monal feces are significantly related to the metabolites we identified; therefore, microbial metabolism is an essential aspect that needs to be evaluated. Microbial biosynthesis of fatty acid may be due to cellular structure or storage, de novo synthesis from glucose, or incorporation of exogenous fatty acids directly into lipid structures [34, 35].
Nutrient levels of commercial pellet feed (as fed basis, %)
The content of metabolites in the feces relating to bile acid biosynthesis and bile secretion had a statistically significant difference between the WCM and CCM. Our results showed differences in metabolism of bile acid, an important component of bile, which plays a role in digestion and metabolism of dietary lipids and cholesterol [36, 37]. Chenodeoxycholate, taurochenodeoxycholate and taurocholate are bile salts that are hydrolyzed into free bile acids by bile salt hydrolase . Chenodeoxycholate amount was higher in the feces of CCM, whereas taurochenodeoxycholate and taurocholate were more abundant in the WCM. Taurodeoxycholic acid and tauroursodeoxycholic acid are taurine conjugated bile acids that were expectedly more abundant in the feces of WCM. The type of bile salt that is most abundant may reflect how the corresponding bile acids play a major role in digestion which is dependent on the dietary composition of each individual bird . In the wild, Chinese monals primarily consume seeds and roots of shrubs in the winter when food is scarce, but they also consume a small amount of moss, earthworms and insect pupa [40, 41]. High concentrations of total non-structural carbohydrate (TNC) reserves are usually found in root tissues of plants [42–44], and may serves as one of the main energy sources for WCM. Because of the seed consumption, the WCM also need to secrete bile acids for fat digestion thus providing another valuable energy source. We also found a high correlation between the bile acid metabolites and gut microbial flora. There exists a mutual regulation between intestinal microbes and bile acids, for microbiome structure appear to be a major regulator of bile acid pool size and composition . Kim G et. al. (2005) found that several bacterial genera in Firmicutes and Actinobacteria can hydrolyze bile salt , among which Enterococcus was significantly related to the above metabolites in this study.
A significantly higher abundance of carbohydrates was found in the feces of WCM as opposed to the CCM, including galactinol, raffinose, stachyose, sucrose, and cellobiose. In plants, raffinose and stachyose are oligosaccharides synthesized from sucrose, subsequently galactitol and galactose are also involved . Animals don’t secrete enzymes that utilize raffinose-series oligosaccharides, which are likely to be digested by microbial enzymes at the end of the gastrointestinal tract . Coon et al. (1990) reported that ileal digestibility of raffinose and stachyose was less than 1.0% in roosters; however, the digestibility, determined with excreta collection, reached 90.5% and 83.8%, respectively . Previous studies have also indicated several Passerine bird species have a low digestibility of sucrose [49, 50] as fecal sugar content increased after consuming sucrose solutions . Cellobiose is an important hydrolytic product of cellulose degradation . Plants contain significant quantities of cellulose, which is difficult to naturally digest by most birds species, but can be utilized by bacteria inhabiting the intestinal tract, especially within the ceca . Yuzhang Wang (2007) reported that the cecum of the Chinese monal is well developed, accounting for 24.09% of the total length of the intestine , which may help the species utilize cellulose and other indigestible components of the diet. Microorganisms in genus that have significant positive correlation with these metabolites may play a big role in these processes.
Indole-containing metabolites were significantly different in fecal samples of CCM, such as 5-hydroxyindoleacetic acid (5-HIAA) and indole-2-carboxylic acid, which are nearly 100 and 300 times higher than that in the wild, respectively. Indole and its derivatives widely exist in animals, microorganisms and plants , and are the main metabolites of serotonin . Degg et. al. (2002) found that excretion of indole containing metabolites, such as 5-HIAA, increased after humans ingested high serotonin-containing foods including tomatoes, bananas, etc. . Our results which indicate higher levels of 5-HIAA and indole-2-carboxylic acid in CCM feces is likely associated with the incorporation of tomatoes in their routine diet. These two altered indole-containing metabolites were also highly correlated with fecal microbial flora. Previous studies have suggested that intestinal bacteria can conversion of tryptophan to indole through enzymatic processing, then form indole-containing metabolites [37, 57]. These studies showed that metabolic processes in intestinal bacteria are essential for the synthesis of indole-containing metabolites, therefore, it may be a specific indicator of intestinal bacteria differences.
Finally, the last metabolite with a significant difference observed between the two groups is sinigrin (2-propenyl glucosinolate). Sinigrin was several thousand times higher in the feces of WCM than that observed in CCM. Sinigrin, which is enriched in plants such as cruciferous vegetables, leaf mustard, and horseradish, has been associated with carbohydrate regulation and lipid metabolism and has also been shown to have anti-neoplasia and anti-microbial properties . In vitro experiments confirmed that sinigrin was degraded by rat intestinal microbiota . Enterococcus and Bacteroides in the human intestine can degrade glucosinolates , and have a significant positive correlation with sinigrin in this study. Therefore, based on our findings of high levels of sinigrin in WCM feces, we can speculate that the diet of WCM also contains a large amount of sinigrin. Although the intestinal flora plays a role in the digestion process, it is more excreted through feces.