Single-cell transcriptomic landscape of the sheep rumen provides insights into physiological programming development and adaptation of digestive strategies.

As an important evolutionary innovation and unique organ, the rumen has played a crucial role in ruminant adaptation to complex ecological environments. However, the cellular basis of its complex morphology and function remains largely unknown. In this study, we identified eight major cell types from seven representative prenatal and postnatal rumen samples using ~56 600 single-cell transcriptomes. We captured the dynamic changes and high heterogeneity in cellular and molecular profiles before, during, and after the appearance of keratinized stratified squamous epithelium with neatly arranged papillae and functional maturity. Basal cells, keratinocytes, differentiating keratinocytes, terminally differentiated keratinocytes, and special spinous cells provided the cellular basis for rumen epithelium formation. Notably, we obtained clear evidence of two keratinization processes involved in early papillogenesis and papillae keratinization and identified TBX3 as a potential marker gene. Importantly, enriched stratum spinosum cells played crucial roles in volatile fatty acid (VFA) metabolism and immune response. Our results provide a comprehensive transcriptional landscape of rumen development at single-cell resolution, as well as valuable insight into the interactions between dietary metabolism and the rumen.

Comparative studies of the composition of bacterial microbiota associated with the ruminal content, ruminal epithelium and in the faeces of lactating dairy cows.

The objective of this research was to compare the composition of bacterial microbiota associated with the ruminal content (RC), ruminal epithelium (RE) and faeces of Holstein dairy cows. The RC, RE and faecal samples were collected from six Holstein dairy cows when the animals were slaughtered. Community compositions of bacterial 16S rRNA genes from RC, RE and faeces were determined using a MiSeq sequencing platform with bacterial-targeting universal primers 338F and 806R. UniFrac analysis revealed that the bacterial communities of RC, RE and faeces were clearly separated from each other. Statistically significant dissimilarities were observed between RC and faeces (P = 0.002), between RC and RE (P = 0.003), and between RE and faeces (P = 0.001). A assignment of sequences to taxa showed that the abundance of the predominant phyla Bacteroidetes was lower in RE than in RC, while a significant higher (P < 0.01) abundance of Proteobacteria was present in RE than in RC. When compared with the RC, the abundance of Firmicutes and Verrucomicrobia was higher in faeces, and RC contained a greater abundance of Bacteroidetes and Tenericutes. A higher proportions of Butyrivibrio and Campylobacter dominated RE as compared to RC. The faecal microbiota was less diverse than RC and dominated by genera Turicibacter and Clostridium. In general, these findings clearly demonstrated the striking compositional differences among RC, RE and faeces, indicating that bacterial communities are specific and adapted to the harbouring environment.

Microbiome-metabolome analysis reveals unhealthy alterations in the composition and metabolism of ruminal microbiota with increasing dietary grain in a goat model.

Currently, knowledge about the impact of high-grain (HG) feeding on rumen microbiota and metabolome is limited. In this study, a combination of the 454 pyrosequencing strategy and the mass spectrometry-based metabolomics technique was applied to investigate the effects of increased dietary grain (0%, 25% and 50% maize grain) on changes in whole ruminal microbiota and their metabolites using goat as a ruminant model. We observed a significant influence of HG feeding in shaping the ruminal bacterial community structure, diversity and composition, with an overall dominance of bacteria of the phylum Firmicutes along with a low abundance of Bacteriodetes in the HG group. High-grain feeding increased the number of ciliate and methanogens, and decreased the density of anaerobic fungi and the richness of the archaeal community. The metabolomics analysis revealed that HG feeding increased the levels of several toxic, inflammatory and unnatural compounds, including endotoxin, tryptamine, tyramine, histamine and phenylacetate. Correlation analysis on the combined datasets revealed some potential relationships between ruminal metabolites and certain microbial species. Information about these relationships may prove useful in either direct (therapeutic) or indirect (dietary) interventions for ruminal disorders due to microbial compositional shifts, such as ruminal acidosis.

High-grain feeding causes strong shifts in ruminal epithelial bacterial community and expression of Toll-like receptor genes in goats.

High-grain (HG) feeding used in intensive goat production can affect the physiology of the rumen wall, but the changes induced in the epimural bacterial community and host Toll-like receptors (TLRs) are not well understood. In this study, 10 male goats were randomly allocated to two groups and fed either a hay diet (0% grain; n = 5) or an HG diet (65% grain; n = 5). The changes in the ruminal epithelial bacterial community and expression of TLRs during long-term (7 weeks) HG feeding were determined using pyrosequencing and quantitative real-time polymerase chain reaction. Principal coordinate analysis and analysis of molecular variance (AMOVA) results showed that HG feeding caused a strong shift in bacterial composition and structure. At the genus level, our data revealed that it increased the relative abundance of taxa Butyrivibrio, unclassified Clostridiales, Mogibacterium, unclassified Anaerolineaceae, and Succiniclasticum, and decreased the proportion of unclassified Ruminococcaceae, unclassified Rikenellaceae, unclassified Erysipelotrichaceae, Howardella, and unclassified Neisseriaceae. The HG-fed goats also exhibited upregulation of the relative mRNA expression of TLR2, TLR3, and TLR5 in the rumen epithelium (P < 0.05). Correlation analysis revealed that the increase in TLR expression was associated with changes in the relative abundance of ruminal epithelial bacteria. This study provides a first insight into the adaptive response of ruminal epithelial bacterial populations to HG feeding in goats and shows that these changes were associated with alterations in TLR expression. These findings provide new insight into understanding of host-microbial relationships in ruminants.

Effects of Acarbose Addition on Ruminal Bacterial Microbiota, Lipopolysaccharide Levels and Fermentation Characteristics In vitro.

This study investigated the effects of acarbose addition on changes in ruminal fermentation characteristics and the composition of the ruminal bacterial community in vitro using batch cultures. Rumen fluid was collected from the rumens of three cannulated Holstein cattle fed forage ad libitum that was supplemented with 6 kg of concentrate. The batch cultures consisted of 8 mL of strained rumen fluid in 40 mL of an anaerobic buffer containing 0.49 g of corn grain, 0.21 g of soybean meal, 0.15 g of alfalfa and 0.15g of Leymus chinensis. Acarbose was added to incubation bottles to achieve final concentrations of 0.1, 0.2, and 0.4 mg/mL. After incubation for 24 h, the addition of acarbose linearly decreased (p<0.05) the total gas production and the concentrations of acetate, propionate, butyrate, total volatile fatty acids, lactate and lipopolysaccharide (LPS). It also linearly increased (p<0.05) the ratio of acetate to propionate, the concentrations of isovalerate, valerate and ammonia-nitrogen and the pH value compared with the control. Pyrosequencing of the 16S rRNA gene showed that the addition of acarbose decreased (p<0.05) the proportion of Firmicutes and Proteobacteria and increased (p<0.05) the percentage of Bacteroidetes, Fibrobacteres, and Synergistetes compared with the control. A principal coordinates analysis plot based on unweighted UniFrac values and molecular variance analysis revealed that the structure of the ruminal bacterial communities in the control was different to that of the ruminal microbiota in the acarbose group. In conclusion, acarbose addition can affect the composition of the ruminal microbial community and may be potentially useful for preventing the occurrence of ruminal acidosis and the accumulation of LPS in the rumen.

A high-grain diet alters the omasal epithelial structure and expression of tight junction proteins in a goat model.

The omasal epithelial barrier plays important roles in maintaining nutrient absorption and immune homeostasis in ruminants. However, little information is currently available about the changes in omasal epithelial barrier function at the structural and molecular levels during feeding of a high-grain (HG) diet. Ten male goats were randomly assigned to two groups, fed either a hay diet (0% grain; n = 5) or HG diet (65% grain; n = 5). Changes in omasal epithelial structure and expression of tight junction (TJ) proteins were determined via electron microscopy and Western blot analysis. After 7 weeks on each diet, omasal contents in the HG group showed significantly lower pH (P <0.001) and significantly higher concentrations of free lipopolysaccharides (LPS; P = 0.001) than the hay group. The goats fed a HG diet showed profound alterations in omasal epithelial structure and TJ proteins, corresponding to depression of thickness of total epithelia, stratum granulosum, and the sum of the stratum spinosum and stratum basale, marked epithelial cellular damage, erosion of intercellular junctions and down-regulation in expression of the TJ proteins, claudin-4 and occludin. The study demonstrates that feeding a HG diet is associated with omasal epithelial cellular damage and changes in expression of TJ proteins. These research findings provide an insight into the possible significance of diet on the omasal epithelial barrier in ruminants.

Discovery of a novel rumen methanogen in the anaerobic fungal culture and its distribution in the rumen as revealed by real-time PCR.

BACKGROUND: The novel archaea belonging to Rumen Cluster C (RCC), which may play an important role in methane production in the rumen have received increased attention. However, the present information on RCC in the rumen is limited by the unsuccessful isolation of axenic pure RCC from the rumen. In the present study, RCC grown in anaerobic fungal subcultures was identified by the molecular and culture methods. RESULTS: A novel RCC species existing in the fungal subcultures was identified and demonstrated by the 16S rRNA gene clone library. Interestingly, the novel RCC species survived in the fungal cultures over all the subculture transferring, even in the 62nd subculture, in contrast to the other methanogens, which disappeared during subcultures. Further work showed that subculture transfer frequency significantly affected the relative abundance of the novel RCC species in the fungal subcultures. The five-day and seven-day transfer frequencies increased the relative abundance of the RCC species (P<0.05). In addition, quantitative real-time PCR revealed that high concentrate diets did not affect the abundance of archaea, but numerically reduced the abundance of the novel RCC species in the rumen. In addition, the relative abundance of the RCC species was numerically higher in the rumen liquid fraction than in the rumen epithelium and solid fractions. Finally, a purified fungal culture containing the RCC species was successfully obtained. PCR and sequencing analysis showed that the novel RCC species contained a mcrA gene, which is known to play a crucial role in methanogenesis, and thus could be identified as a methanogen. CONCLUSION: In this study, a novel RCC species was identified as a methanogen and closely associated with anaerobic fungi. This novel approach by using co-culture with anaerobic fungi may provide a feasible way to culture and investigate not yet identified methanogens.

A high-grain diet causes massive disruption of ruminal epithelial tight junctions in goats.

Alterations in rumen epithelial tight junctions (TJs) at the tissue and molecular levels during high-grain (HG) diet feeding are unknown. Here, 10 male goats were randomly assigned to either a hay diet (0% grain; n = 5) or HG diet group (65% grain; n = 5) to characterize the changes in ruminal epithelial structure and TJ protein expression and localization using scanning and transmission electron microscopy, quantitative real-time PCR, Western blot analysis, and immunofluorescence. After 7 wk of feeding, ruminal free LPS in HG group increased significantly (P < 0.001) compared with the hay group, and free LPS in the peripheral blood was detectable with concentrations of 0.8 ± 0.20 EU/ml, while not detectable in the control, suggesting a leakage of LPS into the blood in the HG group. Correspondingly, the HG-fed goats showed profound alterations in ruminal epithelial structure and TJ proteins, depicted by marked epithelial cellular damage and intercellular junction erosion, down-regulation of TJ proteins claudin-4, occludin, and zonula occludens-1 mRNA and protein expression, as well as redistribution of claudin-1, claudin-4, and occludin. Furthermore, these changes in TJ proteins in the HG group were coupled with the upregulation of mRNA levels for the cytokines TNF-α and IFN-γ in the ruminal epithelia. These results demonstrated for the first time that the HG diet feeding caused disruption of ruminal epithelial TJs that was associated with a local inflammatory response in the rumen epithelium. These findings may provide new insights into understanding the role of TJ proteins in the ruminal epithelial immune homeostasis of ruminants.

Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by (1)H NMR Spectrometry.

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and α-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and α-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.

Effect of Saccharomyces cerevisiae Fermentation Product on Lactation Performance and Lipopolysaccharide Concentration of Dairy Cows.

To evaluate lactation performance and changes in plasma and fecal lipopolysaccharide (LPS) concentrations in response to the supplementation of Saccharomyces cerevisiae fermentation product (SC), two dairy farms were selected. On each farm, 32 cows in early to mid lactation (21 to 140 DIM) were blocked by parity and days in milk (DIM), and randomly assigned to one of the two treatments within block (Control or 56 g SC/cow/d). Effect of SC on lactation performance (daily) and changes in blood and fecal LPS level were examined on d 0 and 28 of supplementation. The results showed that SC supplementation increased lactation performance of dairy cows on both farms. On Farm 1, milk production, 3.5% fat corrected milk (FCM), and yield of milk fat and protein were greater (p<0.01) for cows supplemented with SC. Supplementation of SC increased percentage milk fat (p = 0.029) from 81 to 110 DIM. There was no significant effect (p>0.05) of SC supplementation on percentage of milk protein, dry matter intake and feed efficiency. On Farm 2, cows supplemented with SC had a greater (p<0.05) milk yield, percentage of milk fat and milk protein, yield of milk fat and protein, 3.5% FCM and feed efficiency. Supplemental SC had no effect on LPS concentrations in feces (p>0.05) while it trended to reduce (p = 0.07 or 0.207) the concentration in plasma. The results indicate that supplemental SC can increase lactation performance of dairy cattle and has potential for reducing plasma LPS concentration.

Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane.

This study aimed to obtain natural cultures of anaerobic fungi and their indigenously associated methanogens from herbivores and investigate their ability to degrade lignocelluloses to methane. Eight natural cultures were obtained by Hungate roll tube technique. The fungi were identified as belonging to Piromyces, Anaeromyces and Neocallimastix respectively by microscopy, and the methanogens as Methanobrevibacter spp. by 16S rRNA gene sequencing. In vitro studies with rice straw showed that these cultures degraded 33.5-48.3% substrate and produced 0.33-0.84 mmol/(100ml culture) methane. Two cultures were further selected for their ability to degrade different lignocellulosic materials and could produce 0.38-1.27 mmol/(100ml culture) methane. When methanogens were inhibited, the lignocellulose-degrading ability of cultures significantly reduced. In conclusion, natural cultures of anaerobic fungi with indigenously associated methanogens with high fiber degradation ability were obtained, and these cultures may have the potential in industrial use in lignocelluloses degradation and methane production.

Phylogenetic analysis of methanogens in the pig feces.

In order to assess methanogen diversity in feces of pigs, archaeal 16S rRNA gene clone libraries were constructed from feces of the pig. After the amplification by PCR using primers Met86F and Met1340R, equal quantities of PCR products from each of the five pigs were mixed together and used to construct the library. Sequence analysis showed that the 74 clones were divided into ten phylotypes as defined by RFLP analysis. Phylogenetic analysis showed that three phylotypes were most closely affiliated with the genus Methanobrevibacter (46% of clones). The library comprised 55.4% unidentified euryarchaeal clones. Three phylotypes (LMG4, LMG6, LMG8) were not closely related to any known Euryarchaeota sequences. The phylogenetic analysis indicated that the archaea found in the libraries were all clustered into the Euryarchaeota. The data from the phylogenetic tree showed that those sequences belonged to three monophyletic groups. Phylotypes LGM2 and LGM7 grouped within the genus Methanobrevibacter. Phylotypes LGM4, LGM6, LGM8 and LGM9 grouped within the genus Methanosphaera. Other phylotypes grouped together, and formed a distantly related sister group to Aciduliprofundum boonei and species of the Thermoplasmatales including Thermoplasma volcanium and Thermoplasm acidophilum. Our results showed that methanogens belonging to the genus Methanobrevibacter were predominant in pig feces, and that many unique unknown archaea sequences were also found in the library. Nevertheless, whether these unique sequences represent new taxonomic groups and their role in the pig gut need further investigation.

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle.

Three methanogen 16S rRNA gene clone libraries were constructed from liquid (LM), solid (SM) and epithelium (EM) fractions taken from the rumen of Jinnan cattle in China. After the amplification by PCR using methanogen-specific primers Met86F and Met1340R, equal quantities of PCR products from the same fractions from each of the four cattle were mixed together and used to construct the three libraries. Sequence analysis showed that the 268 LM clones were divided into 35 phylotypes with 18 sequences of phylotypes affiliated with the genus Methanobrevibacter (84.3% of clones). The 135 SM clones were divided into 19 phylotypes with 11 phylotypes affiliated with the genus Methanobrevibacter (77.8%). The 267 EM clones were divided into 33 phylotypes with 15 phylotypes affiliated with the genus Methanobrevibacter (77.2%). Clones closely related to Methanomicrobium mobile and Methanobrevibacter wolinii were only found in the LM library, and those to Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii only in the SM library. LM library comprised 12.4% unidentified euryarchaeal clones, SM library 23.7% and EM library 25.5%, respectively. Five phylotypes (accession number: EF055528 and EF055531-EF055534) did not belong to the Euryarchaeota sequences we had known. One possible new genus (represented by phylotype E17, accession number EF055528) belonging to Methanobacteriaceae was identified from EM library. Quantitative real-time PCR for the first time revealed that epithelium fraction had significantly higher density of methanogens, with methanogenic mcrA gene copies (9.95 log 10 (copies per gram of wet weight)) than solid (9.26, P < 0.01) and the liquid (8.44, P < 0.001). The three clone libraries also appeared different in Shannon index (EM library 2.12, LM library 2.05 and SM library 1.73). Our results showed that there were apparent differences in the methanogenic diversity and abundance in the three different fractions within the rumen of Jinnan cattle, with Methanobrevibacter species predominant in all the three libraries and with epithelium fraction having more unknown species and higher density of methanogens.

[Isolation and identification of a lactate-utilizing, butyrate-producing bacterium and its primary metabolic characteristics].

The distal mammalian gut harbors prodigiously abundant microbes, which provide unique metabolic traits to host. A lactate-utilizing, butyrate-producing bacterium, strain LB01, was isolated from adult swine feces by utilizing modified Hungate technique with rumen liquid-independent YCFA medium supplemented with lactate as the single carbon source. It was an obligate anaerobic, Gram positive bacterium, and could utilize glucose, fructose, maltose and lactate with a large amount of gas products. 16S rRNA sequence analysis revealed that it had the high similarity with members of the genus Megasphaera. The metabolic characteristics of strain LB01 was investigated by using in vitro fermentation system. Lactate at the concentration of 65 mmol/L in YCFA medium was rapidly consumed within 9 hours and was mainly converted to propionate and butyrate after 24h. As the level of acetate declined, the concentration of butyrate rose only in the presence of glucose, suggesting that butyrate could possibly be synthesized by the acetyl CoA: butyryl CoA transferase. When co-cultured with lactic acid bacteria strain K9, strain LB01 evidently reduced the concentration of lactate produced by strain K9 and decelerated the rapid pH drop, finally producing 12.11 mmol/L butyrate and 4.06 mmol/L propionate. The metabolic characteristics that strain LB01 efficiently converts toxic lactate and excessive acetate to butyrate can prevent lactate and acetate accumulation in the large intestine and maintain the slightly acidic environment of the large intestine, consequently revealing that stain LB01 could act as a potential probiotics.

Impact of tiny miRNAs on cancers.

miRNAs are a class of small, approximately 22nt, non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. They play profound and pervasive roles in manipulating gene expression involved in cell development, proliferation and apoptosis in various eukaryotes, which, in theory, could provide an access to many human diseases in theory. Recent evidence demonstrates that aberrant miRNA expression is a hallmark of tumor development, revealing that miRNA genes could function as potential oncogenes and repressors in the human body. miRNAs can affect tumorigenesis mainly by interrupting the cell cycle at the cellular level and by interacting with signaling, oncogenes and with the response to environmental factors at the molecular level. The established miRNA expression signature could be a potent tool to diagnose and treat human cancers in the future.

[The dynamics of microorganism populations and fermentation characters of co-cultures of rumen fungi and cellulolytic bacteria on different substrates].

In vitro co-culture technique and DGGE were used to investigate the dynamics of microorganism populations and fermentation characters of co-cultures of rumen fungi and cellulolytic bacteria at different substrates (concentrate to crude ratio in treatment A to E was all rice straw, 3:7, 5:5, 7:3 and all corn, respectively). The results showed that, compared with Oh, fungal population density at 24h increased in treatment B and C, but decreased in treatment A and D, and no fungi was detected in treatment E; but cellulolytic bacteria population at 24h increased with the increasing of concentrate to crude ratio. At the end of 48h fermentation, no fungi were detected in all treatments; cellulolytic bacteria population increased from treatment A to treatment C, but decreased from treatment D. DGGE results showed that samples collected in treatment A, B and C had similar DGGE patterns with about 11 dominant bands, but dominant bands in treatment D and E decreased markedly compared to treatments A, B and C. With the increasing of concentrate to crude ratio, pH value of the co-culture decreased dramatically (P < 0.05). During the fermentation periods, acetate was the major VFA in co-culture, the acetate to propionate ratio decreased from treatment A to treatment C, but increased from treatment D. With the increasing of concentrate to crude ratio, the total VFA at 48h increased from treatment A to treatment C, and then decreased. At the end of 48h fermentation, CMCase activity and xylanase activity were highest in treatment A. alpha-Amylase activity increased from treatment A to treatment D, but treatment E was the lowest in all treatments.

[Tracking of the development of fecal bacterial community of diarrhea piglets by 16S rDNA techniques].

PCR and denaturing gradient gel electrophoresis (DGGE) were used to track the development of fecal bacterial community of 5 diarrhea piglets. Clone libraries were created from complete 16S rDNA of 42-day fecal sample of piglet 1, 2 and 5. From the library, clones had their V6-V8 regions matched predominant bands on the DGGE gel were then sequenced and their 16S rDNAs subjected to an online similarity search. Results revealed that DGGE profiles from fecal samples of 5 piglets changed form simple to complex, and then returned simple to complex, finally tended to be stable and diverse. DGGE profiles from day 2and 16 fecal samples of 5 piglets were simplest, with high similarity (84%). Their most predominant bands were E. coil. Their Shannon index were 1.38 +/- 0.55 and 1.97 +/- 0.26 respectively. DGGE profiles from day 10 of 5 piglets became quite complex, and had quite low similarity (10%) with DGGE profiles from day 2and 16 fecal samples of 5 piglets. The Shannon index was 2.12 +/- 0.24. E. coil still existed, but it was not the most predominant band. There were significant differences of predominant bands between DGGE profiles from day 27 fecal samples and those from day 35 and 42 fecal samples, with 26.28% and 39.2% of similarity respectively. The predominant bands from day 35 and 42 fecal samples of 5 piglets were tended to be stable, with 55% of similarity. The Shannon index of day 27, day 35 and 42 fecal bacterial communities were 2.19 +/- 0.29, 2.20 +/- 0.17 and 2.14 +/- 0.20 respectively. The fecal bacterial communities of day 42 health piglets were mainly consisted of species of Enterococcus, Streptococcus, Clostridium, Peptostreptococcus and Lactobacillus.

[Detection and diversity analysis of rumen methanogens in the co-cultures with anaerobic fungi].

Rumen methanogen diversity in the co-cultures with anaerobic fungi from goat rumen was analyzed. Mix-cultures of anaerobic fungi and methanogens were obtained from goat rumen using anaerobic fungal medium and the addition of penicillin and streptomycin and then subcultured 62 times by transferring cultures every 3 - 4d. Total DNA from the original rumen fluid and subcultured fungal cultures was used for PCR/DGGE and RFLP analysis. 16S rDNA of clones corresponding to representative OTUs were sequenced. Results showed that the diversity index (Shannon index) of the methanogens generated from DGGE profiles reduced from 1.32 to 0.99 from rumen fluid to fungal culture after 45 subculturing, with the lowest similarity of DGGE profiles at 34.7%. The Shannon index increased from 0.99 to 1.15 from the fungal culture after 45 subculturing to that after 62 subculturing, with the lowest similarity at 89.2% . A total of 5 OTUs were obtained from 69. clones using RFLP analysis and six clones representing the 5 OTUs respectively were sequenced. Of the 5 OTUs, three had their cloned 16S rDNA sequences most closely related to uncultured archaeal symbiont PA202 with the same similarity of 95 %, but had not closely related to any identified culturable methanogen. The rest two OTUs had their cloned 16S rDNA sequences sharing the same closest relative, uncultured rumen methanogen 956, with the same similarity of 97% .Their 16S rDNA sequences of these two OTUs also showed 97% similar to the closest identified culturable methanogen Methanobrevibacter sp. NT7. In conclusion, diverse yet unidentified rumen methanogen species exist in the co-cultures with anaerobic fungi isolated from the goat rumen.