| Golden snub-nosed monkey(Rhinopithecus roxellana)is a unique endangered nonhuman primate species in China,and its species conservation has always been an important work in the field of wildlife protection in China.The study on the composition and function of intestinal microbiota is an important means to understand the health of hosts,and also an important method for the healthy growth and species conservation of wild animals in recent years.Therefore,in this doctoral thesis,Rhinopithecus roxellana was chosen as the object to study the effects of gastrointestinal physiology,diet and living environment on its intestinal microbiota composition and function.These research techniques,including Polymerase chain reaction-denatured gradient gel electrophoresis(PCR-DGGE),quantitative real-time PCR(qPCR),high throughput sequencing based on 16S rDNA amplification,hydrochloric acid insoluble ash method(AIA),gas chromatography technology and nuclear magnetic resonance technology(NMR),were used in this study.The purpose of this study is to explore the key flora and function,provide important basic data for the protection and management of Rhinopithecus roxellana population,especially provide practical guidance for improving the health growth of this species.The specific research contents and results are as follows:1 Host gastrointestinal tract shapes the composition and functional difference of gut microbiota of Rhinopithecus roxellana.Rhinopithecus roxellana had the characteristics of a microflora compatible with the physiological structure of its digestive tract.The digestive tract flora mainly belonged to phylum Firmicutes(38.82%～58.24%)and Bacteroides(13.48%～39.77%).Among them,Prevotellaceae dominated in the foregut,while Ruminococcaceae mainly in the hindgut and faecal.The main differential flora of the digestive tract predominated in the stomach.The stomach of Rhinopithecus roxellana had strong carbohydrate metabolism,mainly involved in GH3,GH 43,GH2,GH 25,GH 13 and GT2 families.The dominant bacteria in the stomach was Prevotella multisaccharivorax belong to Prevotellaceae.The related dominant enzyme families were GT2,GH43,GH51 and GH3.GT2 is the dominant enzyme family in the stomach of captive Rhinopithecus roxellana.The last three enzyme families come from the stomach of wild Rhinopithecus roxellana.It is suggested that the function of glycoside hydrolase was stronger in the microbiota activity of Rhinopithecus roxellana,and there were some differences in the stomach metabolism of this animal from different living environments.2 Living environment shapes the faecal microflora microbiota and functional differences of Rhinopithecus roxellana.Changes in the living environment affected the composition,function and metabolism of the faecal microbiota of Rhinopithecus roxellana.The faecal microbiota were mainly Firmicutes(58.78%～69.49%)and Bacteroidetes(13.98%～21.24%).The abundance of Ruminococcaceae,Verrucomicrobiaceae and Christensenellaceae in the captive faecal was lower,but the faecal of Rhinopithecus roxellana in winter had more beneficial flora.The wild faecal had strong carbohydrate metabolism.The dominant enzyme families were GH43,GH3,GH2,GH13,GT2,and GT4;the dominant flora was Firmicutes bacterium CAG:110 and it was related to the GH13 family.The influence of seasonal changes on microbiota and metabolites was more obvious in wild faecal samples.Acetate,xylose,glucose,propionate,butyrate and glutamate were the main differential metabolites.Wild Rhinopithecus roxellana consumed more fiber diets(bark and bud)in spring and winter,and had more acetate and butyrate,which was related to Akkermansia and Ruminococcaceae_UCG-005.Wild faecal samples consumed more leaves in summer had more glutamate,glucose and xylose.These metabolisms were related to Ruminococcaceae_UCG-014 and Ruminococcaceae_UCG-010.In addition,the main faecal differential metabolites of captive and wild Rhinopithecus roxellana in this experiment were less abundant in autumn.The correlation analysis results of fecal microbiota and metabolome showed that Ruminococcaceae had more complex carbohydrate metabolism activities,while Prevotellaceae was more related to the production of short-chain fatty acids.3 Fiber diet improves bacterial diversity and carbohydrate metabolism in Rhinopithecus roxellana fecal.The microbial diversity,the digestibility of neutral detergent fiber,and the content of acetic acid,propionic acid,and butyric acid in the faecal of Rhinopithecus roxellana increased after ingestion of high-fiber diets,but sub-adult individuals had certain restrictions on the proportion of fiber.The main microbiota in faecal were Firmicutes(35.91%～68.50%)and Bacteroides(19.83%～53.85%).The metabolism of the high-fiber diet group mainly included starch and sucrose metabolism,galactose metabolism,sphingolipid metabolism and other glycan metabolism,and the first two metabolisms belonged to the carbohydrate metabolism.The enzyme families associated with these metabolisms were GH13,GT35 and GH2,of which GH13 had a major role.The microbiota associated with these metabolisms were Ruminococcaceae,Clostridiaceae and Spirochaetaceae.In addition,both Ruminococcaceae and Spirochaetaceae had strong starch and sucrose metabolism functions.And the Ruminococcaceae flora associated with this metabolic activity,included uncultured Ruminococcus sp.,Ruminococcus sp.CAG:177,Ruminococcus sp.CAG:724,and Ruminococcus sp.CAG:624,suggested that the functional redundancy of intestinal flora plays an important role in the intestinal metabolism of Rhinopithecus roxellana.4 The statistical verification of Prevotellaceae and Ruminococcaceae.Prevotellaceae and Ruminococcaceae were dominant in the fecal of non-human primates,including folivore(Rhinopithecus roxellana and Trachypithecus francoisi),omnivorous(Macaca mulatta and Macaca fascicularis)and frugivorous(Nomascus leucogenys).Ruminococcaceae dominated in the hindgut(cecum and colon)and faecal of leaf-eating non-human primates,and it was also the dominant bacteria in the foregut of ruminants(sheep).Prevotellaceae dominated in the stomachs of Rhinopithecus roxellana and Trachypithecus francoisi,and its abundance in the stomach of Rhinopithecus roxellana was higher.The abundant of Prevotellaceae and Ruminococcaceae were lower in the digestive tract of bamboo feeding animal(Ailurus fulgens).In conclusion,this study revealed that Rhinopithecus roxellana was a non-human primate with remarkable fermentation characteristics in the foregut(stomach divided into 4 chambers),and its intestinal metabolism is mainly carbohydrate metabolism.Prevotellaceae can represent the metabolic characteristics of the stomach,and Ruminococcaceae can represent the metabolic characteristics of the hindgut(colon)and faecal.At present,the intestinal metabolism function of Rhinopithecus roxellana raised in the zoo is good,but it can properly increase the proportion of dietary fiber,which has a positive effect on the metabolism of this species in captivity,and its metabolism activity can be understood through the detection of Prevotellaceae and Ruminococcaceae in the faecal. |
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