The Analysis Of Microorganism Diversity And Dominant Bacterial Communities In The Rumen Of Sika Deer (Cervus Nippon)

We applied freeze thawing method with CTAB/SDS/proteinase K and lysozyme method to directlyextract genomic DNA of rumen microorganisms from rumen contents. The results showed that genomicDNA larger than23kb were obtained from rumen contents using two methods. The freeze thawingmethod was much more efficient than lysozyme method, however, lysozyme method was much faster.The bacterial communities in the rumen of sika deer feeding oak leaves based (OL group, animal Aand B) and corn stalk based (CS group, animal C and D) diets were analyzed using16S rRNA geneclone libraries, Denaturing Gradient Gel Electrophoresis (DGGE) and Terminal Restriction FragmentLength Polymorphism (T-RFLP). Overall,239sequences were examined from the two groups using16S rRNA clone libraries,139clones from the OL group were assigned to57operational taxonomicunits (OTUs) and100sequences from the CS group were divided into50OTUs. Prevotella-likesequences belonging to the phylum Bacteriodetes were the dominant bacteria in both groups (97.2%OLand77%CS), and sequences related to Prevotella brevis were present in both groups. However,Prevotella shahii-like, Prevotella veroralis-like, Prevotella albensis-like, and Prevotella salivae-likesequences were more prevalent in the OL group than in the CS group, while Succinivibriodextrinosolvens-like and Prevotella ruminicola-like sequences were more abundant in the CS group.The clustering patterns of DGGE and T-RFLP indicated that the forage affected rumen bacterialcomposition, whereas there was difference between animals in the same group. Sequneces analysis ofDGGE showed that Prevotella spp. were the dominant bacteria in two groups, but the genotypes ofPrevotella spp. were different in two groups. The dominant fibrolytic bacterial in two groups includesClostridium spp. and Eubacterium spp.. T-RFs representing81bp,214bp,272bp and308bp weredominant in OL group,90bp,95bp,175bp,273bp and274bp were predominant in CS group, and161bp,259bp,264bp,266bp and284bp were presented in all animals. It is concluded that Prevotellaspp. was the dominnat bacteria in rumen of sika deer, but the bacterial composition appeared to beaffected by diet.DGGE was applied to indentify the difference of the bacterial diversity in the rumen of sika deer,hostein cow and boer goat.The cluster of DGGE profiles showed that banding patterns clusteredtogether according to host species or diets, indicating that the host species and diets significantlyaffected the rumen bacterial diversity. Overall, a total of53,16,53and30sequences were obtainedfrom hostein cow, boer goat, sika deer feeding corn stalk (CS group) and feeding oak leaves (OL group).The known dominant bacteria in the rumen of hostein cow contained Eubacterium ruminantium,Pseudobutyrivibrio ruminis and Prevotella spp., while Clostridium populeti was the dominant bacteriain the rumen of boer goat. In addition, Succinivibrio dextrinosolvens and Prevotella spp. wereabundantly presented in the rumen of sika deer.We constructed two16S rRNA gene clone libraries from OL and CS groups and determined theconcentration of VFAs in the rumen of sika deer. Overall, we obtained197clone sequences, revealing 146unique phylotypes, which were assigned to36OTUs at the species level (98%identity).Methanogens belonging to the genus Methanobrevibacter were the predominant phylotypesrepresenting83.9%(OL library) and85.9%(CS library) of the clones in the two libraries. Of thephylotypes, Methanobrevibacter millerae was the most abundant species in both libraries, but theproportion of Mbr. millerae related clones in the CS library is higher than in OL library (69.5%and51.4%, respectively). Moreover, Methanobrevibacter wolinii-related clones (32.5%) were alsopredominant in the OL library. Methanobrevibacter smithii-related clones and Methanobrevibacterruminantium-related clones accounted for6.5%and6.6%clones in the CS library, whereas, theseclones were not discovered in the OL library. Methanobrevibacter phylotypes were the predominantmethanogens in rumen of sika deer. Tannin-rich plants may affect the distribution of genusMethanobrevibacter at species level, which may lead to the reduced methane production. The results ofVFAs showed that the concentrations of butyrate and total volatile fatty acids in OL group wassignificantly higher than in CS group (P=0.015and P=0.001). From these results, it is concluded thatMethanobrevibacter spp. was the predominant methanogens in rumen of sika deer, which is differentfrom other animals. Moreover, tannin-rich plants affect the methanogen diversity.