A Comparative Analysis Of The Seasonal Differences In The Intestinal Microbiome Of The Blue Sheep In Helan Mountains(Pseudois Nayaur)

Intestinal microbes play an important role in maintaining the dynamic stability of animal's gastrointestinal tract and helping host adapt to different dietary environments,and are closely related to the host's nutrition,metabolism and immunity.In recent years,due to the increase of blue sheep and environmental constraints,the food for blue sheep in Helan Mountains are not sufficient.When season changes,their food types and nutritional status have undergone major changes.In this study,we used non-invasive sampling technology,microsatellite markers,high-throughput sequencing technology,bioinformatics and other methods to study the intestinal microbial diversity and function of blue sheep in Helan Mountains in winter and summer,and to explore the intestinal microbial diversity of seasonal differences and gender differences for blue sheep,made an in-depth analysis of seasonal differences in function of intestinal microbial and response mechanism of changes in food resources.The results were as follows:1.Individual identification and gender identification:A total of 282 bharal individuals were identified in 344 fecal samples with acceptable quality in winter and summer,including 152 males and 130 females.2.Analysis of the intestinal microbial diversity of bharal:The main phyla of the intestinal microbes of the Helan Mountain bharal are Firmicutes and Bacteroidetes,Firmicutes,Clostridia,Clostridiales and Ruminococcaceae enriched in summer,Spirochaetes,Tenericutes,Lachnospiraceae and Bacteroides enriched in winter.There are significant seasonal and gender differences(P<0.01)in the composition and structure of the gut microbial composition of the Bharal.Seasonal changes are more significant than the changes in intestinal flora caused by gender.There are no significant seasonal differences in gut microbial diversity among females,but males have significant seasonal differences(P<0.05);there is no significant gender difference in gut microbial diversity in winter,and significant gender differences in summer(P<0.01).In summer,there is significant gender differences in Ruminococcaceae(P<0.05),and in winter,there is a significant gender difference in Lachnospiraceae(P<0.05).The Bacteroides phylum has a significant seasonal difference in female bharal(P<0.01),and a significant gender difference in summer(P<0.05).The seasonal differences and gender differences of the intestinal flora diversity and community structure of bharal may be a dynamic response to seasonal changes and changes in food resources.At the same time,bacterial pathogens were also found in the intestinal microbes of the bharal,such as Alistipes,Oscillibacter,Odoribacter.Turicibacter,Streptococcus,Desulfovibrio,and Campylobacter.This study has accumulated basic data for disease prevention and control of Helan mountain bharal.3.Intestinal microbial function analysis:we use function annotation,analysis of function abundance,function difference analysis,and metabolic pathway analysis to explore the function of the intestinal flora of blue sheep.Annotated results based on the KEGG database show that Metabolism has the highest gene abundance,followed by Genetic Information Processing,Cellular Processes,and Environmental Information Processing(Environmental Information Processing).Annotated results based on the eggNOG database show that the gene abundance of Replication,recombination and repair is the highest,followed by Translation,ribosomal structure and biogenesis,and Carbohydrates.Transport and metabolism,Amino acid transport and metabolism.CAZy database annotation results show that Glycoside Hydrolases(GHs)genes have the highest abundance,as are Glycosyl Transferases(GTs)and Carbohydrate-Binding Modules(CBMs)genes.In the CAZy functional difference analysis.there are 84 functional differences in level 2 in winter and summer(P<0.05).A total of 228 enzymes were annotated in winter and summer to participate in the biosynthesis and metabolism of amino acids,mainly by Lachnospiraceae,Clostridiales,Bacteroides and Firmicutes bacterium CAG:110 and other flora encoding enzyme genes,among them,43 enzymes have significant seasonal differences.Metabolic pathway analysis results show that the unique metabolic pathways of the intestinal flora of bharal in winter are K03476,K00855,K18337,K19548,K06123,K05363,K01589,K01466 and K06447,and the unique metabolic pathways of the intestinal flora of bharal are K01563,K01607,K10218,K05711,K20454,K20451,K00376,K18118 and K05362.There are 12 metabolic pathways that differ significantly between the winter and summer groups(P<0.05).The metabolic pathways enriched in the summer group are K03088,K06147,K05349,K02355,K01190,K03046,K03043,K01006,K03737,K03497,and K02469,of which 6 metabolic pathways are in the genetic information processing showed high abundance,and the short-chain fatty acid(SCFAs)production pathway was significantly enriched in the summer group;the metabolic pathway enriched in the winter group was K07133.The carbohydrate metabolism function in summer was significantly higher than that in winter(P<0.05).The seasonal differences in the functions of the intestinal flora are beneficial to the bharal adapting to the seasonal changes of the Helan Mountain habitat,changes in food resources,accumulating energy and improving immunity.Based on the above research results,we believe that seasons,sex and food changes will affect the intestinal microbial diversity of the Helan Mountain bharal,and the dynamic changes and functions of the intestinal flora will respond to this change.It plays an important role in the nutrition,metabolism and immunity of bharal.This study not only provides a scientific basis for the physiological and ecological management of bharal in Helan Mountain,the rational and effective use of bharal resources,and the management decision-making in the reserve,but also for the in-depth study of the digestive and physiological mechanisms of cloven-hoofed animals,and the interaction of intestinal flora and the host.The relationship and accumulation of data on the prevention and control of animal digestive tract diseases have important theoretical and practical significance for the protection and utilization of wild animals.