The Effects Of Forage Type And Season On The Microbiota Of Muskoxen Rumen Through Metatranscriptomic Approaches

Muskox?Ovibos moschatus?,as the biggest herbivore in the High Arctic,has evolved to ingest and digest high lignified forages mainly by the symbiotic microbial community in rumen.Triticale straw and brome hay were two cool season crop residues and representatives of different quality forage.During the high intakes period of fall and low intakes period of spring,the microbiota in muskoxen rumen would be variable.So,through a comparative metatranscriptomic approach based on total RNA sequencing,this study investigated the effects of forage type and season on the structure and composition of the global active microbial community including prokaryotes and eukaryotes,the relative abundance of individual phylotype,as well as the synergistic interacted consortia networks in muskoxen rumen.Besides those,the expression of glycoside hydrolase families in the degradation of triticale straw in muskoxen rumen during the period of high intakes duringfall was also studied through a metatranscriptomic approach based on RiboZero RNA sequencing.Then comprehensive insights into the relationship among glycoside hydrolase families,microbial phylotypes,and the nutritional composition of triticale straw were obtained and the putative mechanisms behind were explored.This study included two animal feeding experiments,conducted during August and September in autumn and April and May in spring.Each experiment was carried out under a crossover design.In detail,eight castrated and ruminally-cannulated muskoxen were housed in individual pens and blocked into two groups according to their initial body mass.Each group was crossover fed triticale straw and brome hay during two periods of two consecutive.Each period comprised 3 weeks of adaptation to forage followed by one week of study.The diet comprised concentrate and roughage.Concentrate was supplied to each animal with equal dose of 335 gd^-1for each period,while roughage was supplied to each animal with either triticale straw or brome hay ad libitum.Ruminal solid contents were sampled in the morning feeding on day 4 of study week for each period.Total RNA was isolated from the solid contents and sequenced to decipher the structure and composition of microbiota.RiboZero RNA was purified from total RNA of solid content of muskoxen rumen fed triticale straw during fall and sequenced to analyze the expression of protein coding genes,especially the glycoside hydrolase families.The main findings are as follows:In the microbiota of muskoxen rumen,bacteria and eukaryotes totally accounted for 99%and approaximated to a ratio of 1:1,however,forage type had no effect on the abundance of bacteria and eukaryotes?P>0.05?;while archaea account for 1%.Forage type and season significantly affected the relative abundance of archaea?P<0.05?,which was more in the muskoxen rumen fed triticale straw than brome hay,and during spring than fall,however,while without effect on those of bacteria and eukaryotes?P<0.05?.The main phylotypes of lignocellolytic and fermenting fermentable substrates such as Fibrobacter,Ruminococcus,and Neocallimastix,as well as Prevotella were the predominant in muskoxen rumen fed triticale straw and brome hay during fall and spring,respectively.Between-class principal component analysis?PCA?revealed that there were significant differences in the structures and compositions of bacteria and archaea between muskoxen rumen fed different forage and the same results were observed during both fall and spring?P<0.05?;while the structures and compositions of fungi and protozoa were both of significant difference during fall?P<0.05?and not significant during spring?P>0.05?.There were no significant differences in the structures and compositions of fungi and protozoa between muskoxen rumen fed during fall and spring and the same results were observed both when animals fed triticale straw and brome hay?P>0.05?;while the structures and compositions of bacteria and archaea were both of significant difference when animals fed triticale straw?P<0.05?and not significant when fed brome hay?P>0.05?.LEFSe analysis results showed that when degrading and utilizing different forage,the muskoxen rumen selected different lignocellulolytic,soluble substances fermenting,and hydrogen scavenging and energy transferring phylotypes to act.When grazing triticale straw during fall,muskoxen rumen selective enriched 10lignocellulytic phylotypes such as Cyllamyces,9 fermenting phylotypes such as Rikenellaceae,and 17 hydrogen-scavenging and energy-transferring phylotypes such as Methanomicrobium;when grzing brome hay,the selective enriched phylotypes were 8,13,and 10,respectively.The effect of forage on the microbial phylotypes depended the season.The number of phyotypes of lignocellulolytic,soluble substances fermenting,and hydrogen scavenging and energy transferring selected selected by triticale strawwere more during fall than spring,which were 10,9,and 17during fall,and 6,5,6 during spring,respectively.However,6 lignocellulolytic phylotypes,such as Ruminococcus₂,2 fermenting phylotypes,such as Rikenellaceae,and 5 phylotypes of scavenging hydrogens and delivering energy,such as Methanomicrobium,were selectively enriched by triticale straw and all were enriched during fall and spring.The results of Pearson correlation analysis showed that the synergistic interaction and consortia network interacted by the phylotypes of microbiota in muskoxen rumen varied according to the season and forage type,which were more in muskoxen rumen fed triticale straw than brome hay during fall,while similar during spring;and more during fall than spring when animals fed triticale straw,while just the opposite when animals fed brome hay.In the degradation of triticale straw in muskoxen rumen,glycoside hydrolase families mainly degraded celluloses,hemicelluloses,and oligosaccharides.Families of GH48,GH45,GH5,GH9,GH6,and GH94 were the main cellulolytic families,and mainly originated from Fibrobacter,Ruminococcus,Neocallimastix,Piromyces,Polyplastron,Epidinium.Families of GH11,GH10,GH74,GH30,GH8,GH26,and GH16 were the main hemicellulolytic families,and mainly produced from Fibrobacter,Ruminococcus,Neocallimastix,Piromyces,Orpinomyces,Polyplastron and Epidinium.Families of GH43,GH1,GH3,GH33,and GH109 were the main oligosaccharides degrading families and mainly produced from Prevotella,Butyrivibrio,Cellulosilyticum,Neocallimastix,and Piromyces.Conclusion:In the microbiota of muskoxen rumen,eukaryotes accounted for high proportion which was approximated to equal to that of bacteria;while archaea account for 1%,which was higher in muskoxen rumen fed triticale straw than brome hay and during spring than fall.the effectofseason on the structure and composition of microbial community was larger in muskoxen rumen fed triticale straw than brome hay.Muskoxen rumen selectively enriched more lignocellulolytic and hydrogen reduction of energy transfering phylotypes when fed triticale straw than brome hay,and during fall than spring;while less fermentable substrate fermenting phylotypes when animals fed triticale straw than brome hay,and during spring than fall.The potential of phylotypes such as Ruminococcus,Neocallimastix,and Fibrobacter to carry out the biochemical process of degradation of plant fiber were evidenced to be accordant with the metabolic activities themselves in the degradation of triticale straw in muskoxen rumen.The degradation of triticale straw was carried out by the glycoside hydrolase families of GH48,GH45,GH11,GH10,GH43,and GH1 produced by the dominant phylotypes,phylotypes selectively enriched by triticale straw and the synergistic consortia networks interacted by those phylotypes in muskoxen rumen during fall.