| Rumen microbial fermentation can convert human-inedible,low-quality plant lignocelluloses into high-quality protein foods,such as milk and meat,thus making ruminant production a great contribution to food security.Rumen fermentation relies on the microorganisms residing in the rumen,including bacteria,archaea,fungi,and protozoa.Therefore,manipulation of the rumen microbial composition to improve the rumen function is an effective way to improve ruminant production.Deciphering the relationship between the complex composition of rumen microbes and their functions can provide a more scientific and reasonable direction and goal for regulating rumen function.This study compared differences in the composition and function of rumen microbes in different types of diets and animal species(bison and bull)using metagenomics technology and then explored the main microbial species that highly correlated to differential microbial functions that can improve rumen fermentation.The in vitro fermentation experiment of thymol regulating the microbial composition of the rumen was conducted to further explored the correlation between changes in rumen microbial composition and rumen fermentation functions.The main research contents and results are as follows:Part ? Establishment of Kraken2 method and its application verification in rumen microbial taxonomy analysisThis study established the Kraken2 database specially for rumen microbial composition analysis based on complete genomes of bacteria and archaea in the NCBI RefSeq database,and the latest metagenome-assembled genomes of rumen bacteria and archaea.A previous published metatranscriptomic dataset using the Kraken method was adopted for the validation of the Kraken2 method as well as comparative analysis between the newly established Kraken2 method and the Kraken method in the taxonomy analysis.In the metatranscriptomic dataset,animals were divided into two groups,6 head of Angus bulls for either low feed efficiency(LFE)and high feed efficiency(HFE),main results for this experiment are as follows:(1)The rate of sequence annotated in Kraken2 was 72.79%± 11.01%,which was much higher than that of Kraken's 31.50%±6.74%,indicated that the newly established Kraken2 method classified more sequences compared with the previous Kraken method.(2)In bacterial composition,little difference was found between Kraken2 and Kraken method,similar bacterial composition was obtained at either phylum or genus level.The genera Ruminiclostridium and Xenorhabdus were exclusively detected in the Kraken method,and the relative abundance of Xenorhabdus was significantly lower(FDR<0.15)in the high feed efficiency group compared with the low feed efficiency group.The genera Christensenella(1.08%)and Faecalibacterium(0.27%)were exclusively detected in the Kraken2 method,and Faecalibacterium was more abundant(FDR<0.15)in the high feed efficiency group.(3)In archaeal composition,the results of the Kraken2 method differed a lot from Kraken in dominant taxa at different taxonomic levels.The genus Candidatus Methanomethylophilus(16.02%)was exclusively detected in Kraken,however,Kraken2 detected several genera not identified in Kraken,including Thermococcus,Methanosphaera,and other five genera.(4)Beta diversity analysis showed that,whether in Kraken2 or Kraken,rumen bacterial or archaeal communities with different feed efficiency were not significantly different(P>0.05).A trend(P=0.092)to shift was observed in the communities of archaea with different feed efficiency in Kraken2.Part ? summary:The newly established Kraken2 method has greatly improved the accuracy of archaeal taxonomy analysis compared with the previous Kraken method,therefore,the newly established Kraken2 method has great advantages in the latter study.Part II Metagenomics exploring of rumen microbial composition and function characteristicsTwo species of ruminants,the American bison and the Angus bull were adopted in this experiment,and both were divided into two groups fed with background diet(BCK diet)and high grain diet(HG diet),respectively.Either group consists of 8 head of animals.Metagenomic sequencing technology was employed to analyze the rumen microbial composition and functional composition in SEED subsystems,as well as the distribution of functional genes in CAZymes(Carbohydrate-active enzymes).The taxonomy and functional profiles were connected by the same sequence IDs annotated in different databases,and the significant correlations between the differential functions(including differential CAZymes)with the microbial taxa were established by Spearman correlation analysis.The main results are as follows:(1)Except for fourteen bacterial phyla commonly shared by the bison and bull,the phylum Tenericutes(0.58%)was exclusively detected in the bull.Only the relative abundance of Fibrobacteres was significantly higher(FDR<0.05)in the bull fed with BCK diet compared with HG diet,other phyla did not show a significant difference in the relative abundance between diet types or animal species(FDR<0.05).Both in the bison and bull,the Selenomonas and Sarcina were enriched in the HG diet and BCK diet,respectively.Besides,the relative abundance of Fibrobacter,Alistipes,and the other three genera were significantly different between BCK and HG diets in the bull(FDR<0.05).The bison and bull had little difference in bacterial composition when both animal species fed with BCK diet,while the relative abundance of Olsenella,Desulfovivrio,and Megasphaera were significantly higher(FDR<0.05)in the bull compared to the bison both fed with HG diet.(2)At the genus level of rumen archaea,only Candidatus Methanomethylophilus(0.17%)was exclusively detected in the bison rumen,and main genera(relative abundance ? 0.5%)in both the bison and bull did not differ between different types of diets or different animal species.At the species level,only the relative abundance of methanogenic archaeon ISO4-H5(0.71%)and uncultured euryarchaeote(2.03%)in the bull fed with HG diet was significantly higher than that of bulls fed with BCK diet(FDR<0.05).When both the bison and bull fed with BCK diet,the relative abundance of Methanobrevibacter millerae,Methanomassiliicoccaceae archaeon DOK,and Candidatus Methanomethylophilus alvus were significantly higher in the bison compared with the bull(FDR<0.05).Whether in BCK or HG diet,uncultured euryarchaeote had a higher relative abundance in the bull compared with the bison(FDR<0.05).(3)The diet type exerted less effect on the functional composition of the bison in SEED subsystems compared with the bull.In the bison,a little level3 functional categories belonging to carbohydrates,cofactors,vitamins,prosthetic groups,and pigments,and protein metabolism at level 1 were significantly enriched(P<0.05)in the BCK diet group compared with the HG diet group,while several functional categories at level3 belonging to protein metabolism,carbohydrates,and amino acids and derivatives were significantly abundant(P<0.05)in HG diet compared with BCK diet.Compared with the bison,many more functional categories were found significantly enriched in the bulls fed with HG compared with the BCK diet group,including functions belonging to amino acids and derivatives,carbohydrates,protein metabolism,and cofactors,vitamins,prosthetic groups,and pigments.The bison had more enriched functional categories at level3 compared with the bulls either in BCK or HG diet.In the same BCK diet,the functional categories at level3 enriched(P<0.05)in the bison belonged to carbohydrates,amino acids and derives,cofactors,vitamins,prosthetic groups,pigments,while functions belonging to fatty acids,lipids,and isoprenoids were more abundant(P<0.05)in the bull.Similar to the BCK diet,in the same HG diet,more functional categories at level3 were enriched(P<0.05)in the bison compared with bulls,the functional categories belonged to carbohydrates,cofactors,vitamins,prosthetic groups,pigments,and fatty acids,lipids,and isoprenoids,while functions belonging to protein metabolism were more abundant in the bull compared with the bison.(4)The diet type exerted a greater effect on the composition of rumen microbial functional genes encoding CAZymes in the bull compared with the bison,many more differentially abundant CAZymes were observed in the bull.In both the bison and the bull,GHs and CBMs related to cellulose and hemicellulose degradation were significantly enriched in the BCK diet,while GHs and CBMs related to oligosaccharides degradation,and the glycosyltransferase(GTs)were significantly enriched in the HG diet.In the same BCK diet,GH113 and CBM61 related to the hemicellulose degrading were significantly enriched in the bison compared with bulls,and the enriched CAZymes in the bulls were CBM26,GH24,GH73 participating in the degradation of oligosaccharides.In the same HG diet,GHs and CBMs related to the cellulose or hemicellulose degrading had higher abundance in the bison compared with the bull,while highly abundant in the bull were GHs or CBMs responsible for the oligosaccharide degrading.(5)Some microbial taxa were functionally classified in this study.The bacterial genera of Acidaminococcus and Dialister were positively correlated(p>0.7 and adjusted P<0.001)with methane production related functions,while Brachyspira,Olsenella and Megasphaera were found to be negatively correlated(p<-0.7 and adjusted P<0.001)with methane production related function.The bacterial genus of Alistipes was positively correlated with vitamin B production in the rumen.In terms of amino acids biosynthesis,the bacterial genus Lachnoclostridium,Blautia was positively correlated with amino acids biosynthesis.Besides,the Prevotella was positively correlated with protein degrading.(6)The bacterial genera of Acidaminococcus,Blautia,Lachnoclostridium,and Roseburia were significantly negatively correlated with several CBMs participating in cellulose and hemicelluloses degradation(such as CBM1,CBM6,CBM10).The Blautia,Aminipila were negatively correlated with glycoside hydrolases responsible for the cellulose or hemicellulose degrading including GH45 and GH5.Besides,the bacterial genera of Blautia,Anaerostipes,Lachnoclostridium,and Roseburia were positively correlated with oligosaccharides degrading related glycoside hydrolases including GH1 and GH4.The bacterial genera of Alistipes,Blautia,Lachnoclostridium,Anaerostipes,Roseburia were positively correlated with different carbohydrate esterases.Part ? summary:The diet type exerts a little effect on rumen microbial composition and functional profiles in the bison,while greatly affects that of the bull.And the bison rumen microbial composition and functional profiles also differ from the bull in the same type of diet.The differences in microbial composition and functional profiles in the bison and bull fed with different diets suggest that the bison is more efficient in the utilization of the forage in the diet,and the bison and bull have different ways of utilising the high concentrate diet.Some rumen microbial taxa are highly correlated with rumen microbial functions affecting rumen fermentation,the regulation of these microbial taxa could improve the rumen fermentation function.Part ? Study of thymol supplementation affecting rumen fermentation functions in vitroIn the second part,we found that eight bacterial genera including Blautia,Lachnoclostridium,and Anaerostipes were highly correlated with the rumen microbial functions affecting fermentation.Thymol is a bioactive compound of essential oils with antimicrobial properties and is likely to affect the rumen microbial composition,thus improve rumen fermentation function.Therefore,the in vitro fermentation experiment was conducted by supplementation of exogenous additives with different concentrations of thymol(0,100 mg/L,200 mg/L,400 mg/L),to explore the effects of thymol on the composition of rumen bacteria,archaea,and protozoa,and rumen fermentation functions.By establishing the correlation between the composition change of the rumen microbes and rumen fermentation products,the main microbial taxa that could improve rumen fermentation were obtained.The main results are as follows:(1)The medium concentration(200 mg/L)or high concentration(400 mg/L)significantly decreased(P<0.05)the methane production.A high concentration of thymol could inhibit the rumen fermentation by significantly decreased(P<0.05)the total gas production,IVDMD,and IVOMD,and significantly increase the ratio of acetate and propionate(P<0.05).Medium or high concentrate of thymol supplementation significantly decreased the production of isobutyrate and valerate.(2)Thymol supplementation exerted a significant impact on the rumen bacterial composition.At the phylum level,medium or high concentration of thymol supplementation dramatically decreased(P<0.05)the relative abundance of Bacteroidetes,and simultaneously increased(P<0.05)the relative abundance of Firmicutes.At the genus level,medium or high concentration of thymol supplementation significantly decreased(P<0.05)the relative abundance of Prevotella,Veillonellaceae UCG-001,and significantly increased(P<0.05)the relative abundance of genera including Streptococcus and Pseudobutyrivibrio.(3)Thymol exerted less impact on rumen archaeal composition compared with bacteria.At the species level,compared with the control group,medium or high concentration of thymol supplementation significantly decreased the relative abundance of unclassified_f_Methanomassiliicoccaceae(P<0.05),and unclassified_c_Methanomicrobia was only significantly increased(P<0.05)by the high concentration supplementation of thymol.(4)The impact of thymol on rumen protozoan composition was even less apparent compared with rumen archaea.Whether at the family level or genus level,the protozoan taxa did not differ(P>0.05)in relative abundance between different concentrations of thymol supplementation groups and the control group.(5)According to the Spearman correlation analysis between rumen microbes(bacteria,archaea,and protozoa)and rumen fermentation products,bacterial genera of Lactobacillus,Pseudobutyrivibrio,Streptococcus,Ruminococcaceae V9D2013 group,Ruminococcaceae UCG-002,Succinivibrio,unclassified_f Prevotellaceae,and Desulfovivrio,and protozoan genus of Isotricha were highly correlated(p>0.7 and adjusted P<0.005)with methane production and VFA production.Part ? summary:200 mg/L of thymol only affects few microbes and could improve rumen fermentation to some extent,while 400 mg/L of thymol can affect the composition of many microbes and inhibit rumen fermentation.This experiment mainly discovered some microbes that could improve rumen fermentation by the methane mitigation and the improvement of VFA production.In summary,the main results of this study are as follows:1.The newly established rumen microbial reference database together with the Kraken2 method,not only improves the efficiency of taxonomy analysis from the optimization of algorithms but also improves the accuracy of taxonomy analysis from the improvement of the database,thus the new Kraken2 method is more reliable and efficient for taxonomy analysis in metagenomics.2.The diet type has a little effect on the bison's rumen microbial composition and functional profiles,while significantly affect that of the bull.The rumen microbial composition and functional profiles of the bison differ from the bull fed with the same type of diets.The differences in the rumen microbial composition and functional profiles in the bison and bull fed with different types of diets suggest that the bison is more efficient in utilising the forage in the diet,and the bison and bull have different ways in the utilisation of high concentrate diet.3.Supplementation with 200 mg/L of thymol can decrease rumen methane production and has no distinctly detrimental impact on rumen bacteria,archaea and protozoa,and the rumen fermentation maintains a normal condition.However,a high concentration of 400 mg/L of thymol significantly inhibits rumen microbiota and rumen fermentation.4.Some special microbial taxa in rumen greatly affect rumen fermentation functions,and the rumen fermentation can be improved by properly regulating the abundance of these microbes... |
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