In the present study, two 16S rRNA gene libraries and PCR-DGGE were used to study the rumen bacteria in the rumen of domesticated Sika deer feeding on oak leaves-based (OL) and corn stalks-based (CS) diets. Sequences from the two clone libraries and PCR-DGGE bands indicated that the majority of sequences belonged to phylum Bacteroidetes. The findings from the current study are similar to previous findings for other ruminants, such as Reindeer, yaks, cattle and goats [14–18]. The predominance of sequences belonging to the phylum Bacteroidetes highlights their important role in the rumen fermentation of domesticated Sika deer. While, the phylum Firmicutes being prevalent in other ruminants were not found in the OL library , which could be caused by the tannins contained in oak leaves, because some studies reported that the tannins in oak leaves may have a negative effect on some of the bacterial species [20, 21], and the growth of proteolytic bacteria, such as Butyrivibrio fibrisolvens, Ruminococcus albus and Streptococcus bovis, were inhibited by tannins [22, 23]. This may also indicate that some species belonging to phylum Firmicutes in the rumen of domestic Sika deer may be sensitive to tannins.
Within the phylum Bacteroidetes, Prevotella-like clones accounted for 97.2% of the clones in the OL group and 77% in the CS group. Moreover, the PCR-DGGE results also showed the genus Prevotella represented the predominant bacteria in rumen of domesticated Sika deer (Table 3), which is in agreement with other studies [19, 24–28] . The prevalence of Prevotella spp. in rumen fermentation of domesticated Sika deer was likely because they utilize a wide variety of polysaccharides, and are thought to be important contributors to xylan degradation in the rumen [29–32]. Although other studies found that concentrate diets increased the numbers of clones related to Prevotella spp. [33, 34], however, in comparison with other ruminants, there was an apparent difference in the proportion of Prevotella spp. [6, 25, 27, 28]. Prevotella spp. belonged to the hydrogen-consuming bacteria, which could produce propionate via succinate or acrylate pathways though fermentation of sugars and lactate, respectively [35–37]. Therefore, the dominant genus Prevotella in the rumen of domesticated Sika deer suggested that the propionate pathway may be relatively vital in the rumen fermentation of domestic Sika deer, which, in turn, may lead to the decreased production of methane, since the succinate-propionate pathway could compete with methanogens for hydrogen . The relationship between Prevotella spp. and methanogens in the rumen of domesticated Sika deer was worth of further investigating. In addition, the bacterial communities in the rumen between domesticated Sika deer, Svalbard reindeer and Norwegian reindeer, all cervids, were compared using Fast UniFrac, which can be used to determine whether communities are significantly different . The results of Principal coordinate analysis (PCoA) between domesticated Sika deer and Reindeer using the Fast Unifrac platform clearly showed that the rumen bacterial communities were distinct, which can be attributed to the host-species (Figure 5) [13, 26, 39].
It is important to note, that fibrolytic bacteria, such as C. populeti, E. cellulosolvens and Ps. ruminis were discovered in our analysis based on PCR-DGGE, rather than the predominant fibrolytic bacteria, B. fibrisolvens, Fibrobacter succinogenes, Ruminococcus flavefaciens and R. albus. This may suggest that the rumen of domesticated Sika deer depend on unique bacterial communities in rumen fermentation. In contrast, the absence of R. flavefaciens, B. fibrisolvens, F. succinogenes and R. albus in the present work may be attributed to the small number of clones may have missed some other members of the bacterial community, and the weak or unidentifiable bands in DGGE. Future work will employ next generation sequencing to effectively elucidate the bacterial diversity present in the rumen of domesticated Sika deer and other livestock. Collectively, these data indicated that the rumen of domesticated Sika deer harbored unique bacterial populations for the fermentation of plant biomass and concentrate diet.
Interestingly, in both clone libraries, none of the sequences were 100% identical. Rather, most clones were in the range of 83-98% identify to known species in both libraries. These results suggested that the rumen bacteria of domesticated Sika deer were not previously characterized and that these clones related to Prevotella spp. in the rumen represented new species. This agrees with previous findings suggesting that most of the bacterial species in rumen of other cervids (96% for Hokkaido Sika deer and 100% for Svalbard reindeer) are unknown [26, 40]. Despite the diets and geographic location are important factors affecting bacterial diversity in the rumen, however, the presence of these unknown or unidentified species may be the result of co-evolution between microbial communities and the host.
PCR-DGGE analysis showed that the bacterial diversity in domesticated Sika deer fed corn stalks differed from the domesticated Sika deer consuming oak leaves (Figure 5), indicating forage affected the relative abundance and composition of the bacteria. Moreover, the difference in the Prevotella species between the two groups was very apparent (Table 3). For instance, the results of clone library showed that the proportion of P. ruminicola-like clones (27%) was abundant in the CS group comparing with those in the OL group, and sequences analysis of PCR-DGGE also indicated that P. ruminicola was only presented in CS group. Interestingly, Prevotella species in the rumen could contribute to cell wall degradation through synergistic interactions with species of cellulolytic bacteria . Therefore, considering the relatively high fiber content (about 36%) in corn stalks, these P. ruminicola-like clones in the CS group may play a role in the degradation of cellulose. This explanation is partly supported by recent metagenomics data from the Svalbard reindeer rumen microbiome, where the presence of polysaccharide utilizing glycoside hydrolase and other carbohydrate-active enzyme families target various polysaccharides including cellulose, xylan and pectin .
In the OL group, the distribution of P. shahii-like clones (16.5%), P. veroralis-like clones (23.8%) and P. salivae-like clones (12.3%) were several times higher in the OL library than in the CS library, and several bands in the PCR-DGGE analysis showed sequence similarities to P. salivae (Table 3). Previous study reported that P. ruminicola may tolerate condensed tannins . Considering the genetic diversity of Prevotella spp. [27, 42], it is assumed that the tolerance to tannins of domestic Sika deer may be related to the abundance of Prevotella spp. in the OL group. In addition, we found two bands (O-3 and O-18) were identified as St. pasteurianus using PCR-DGGE. Thus this species may also be important in the process of degrading tannins in diets, because tannin-degrading capability of Streptococcus sp. have been demonstrated in other studies [43–46]. However, these assumptions need to be investigated in future studies.
Phylogenetic analysis indicated the presence of diet-specific subpopulations of Prevotella. Prevotella clusters 1 and 2 not only demonstrated the genetic diversity of Prevotella spp., but also confirmed the above assumption that clones grouped within clusters 1 or 2 may be related to the degradation of fiber (cluster 1) or tannins (cluster 2), whereas, the clones in cluster 3 may have common features of degrading starch and proteins contained in concentrate diets (Figure 3). However, clones related to the bacterial genera Sporanaerobacter, Parabacteroides and Proteiniphilum were found in the rumen of domesticated Sika deer fed corn stalks that were not previously reported in the rumen from other ruminants. Sporanaerobacter acetigenes is an acetogenic and a sulfur-reducing bacterium that was isolated from an anaerobic sludge blanket reactor in Mexico [47, 48]. The rumen has considerable capacity to convert sulfate into sulfur-containing amino acids. Similarly, little is known about Proteiniphilum acetatigenes, which was originally isolated from a UASB reactor treating brewery wastewater in China . These bacteria in rumen of domesticated Sika deer may have other biological functions and is worthy of further investigation.