Molecular diversity of bacteria in Yunnan yellow cattle (Bos taurs) from Nujiang region, China

The rumen content of four Yunnan Yellow Cattle (Bos taurs) were collected to determine the bacteria diversity by using 16S rRNA gene sequence analysis. A total of 129 sequences were examined and the sequences were referred as 107 OTU (Operational Taxonomy Unit) according to the similarity level of 97% in gene sequence. Similarity analysis revealed that Yunnan Yellow Cattle had 12 sequences (10 OTU) shared 97% or greater similarity with cultured rumen bacteria Butyrivibrio fibrisolvens, Succiniclasticum ruminis, Ruminococcus bromii, Clostridium proteoclasticum, Ruminococcus flavefaciens, Pseudobutyrivibrio ruminis, Jeotgalicoccus psychrophilus, and Prevotella ruminicola, which accounting for 9.3% of the total clones (9.2% of the total OTU). The further 12 sequences (9 OTU) shared 90–97% similarity with cultured bacteria Clostridium aminobutyricum, butyrate-producing bacterium, Schwartzia succinivorans, Prevotella ruminicola, Eubacterium ruminantium, Ruminococcus albus, and Clostridium termitidis, also accounting for 9.3% of the total sequences (8.3% of the total OTU). The remaining 105 sequences (90 OTU) shared less than 90% similarity with cultured bacteria, accounting for 81.4% of the total sequences (82.5% of the total OTU). According to the phylogenetic analysis, all sequences were phylogenetically placed within phyla of low G+C subdivision (accounting for 72.1 and 72.5% of the total clones and OTU, respectively) and CFB subdivision (Cytophaga-Flexibacter-Bacteroides; accounting for 27.9 and 27.5% of the total clones and OTU, respectively). Among the examined clones, rare bacteria Jeotgalicoccus psychrophilus was detected in the rumen of cattle.     

<Emphasis Type="Italic">Dasytricha</Emphasis> Dominance in Surti Buffalo Rumen Revealed by 18S rRNA Sequences and Real-Time PCR Assay

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR–RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10⁵ vs. 1.3 × 10⁴) in per ml ruminal fluid.     

Dominant bacterial communities in the rumen of Gayals (Bos frontalis), Yaks (Bos grunniens) and Yunnan Yellow Cattle (Bos taurs) revealed by denaturing gradient gel electrophoresis

The dominant rumen bacteria in Gayals, Yaks and Yunnan Yellow Cattle were investigated using PCR-DGGE approach. The analysis of DGGE profiles, identification of dominant bands and phylogenetic analysis 16S rDNA sequences in DGGE profiles were combined to reveal the dominant bacterial communities and compared the differences between those cattle species. DGGE profiles revealed that Gayals had the most abundant dominant bacteria and the lowest similarity of intraspecies between individuals than other two cattle species. A total of 45 sequences were examined and sequence similarity analysis revealed that Gayals had the most sequences appeared to uncultured bacteria, accounting for 85.0% of the total sequences, Yaks and Yunnan Yellow Cattle had 44.4 and 68.8% uncultured bacterial sequences, respectively. According to phylogenetic analysis, the rumen dominant bacteria of Gayals were mainly phylogenetically placed within phyla firmicutes and bacteroidetes, and the known bacteria were mainly belonged to the genera Lachnospiraceae bacterium, Ruminococcus flavefaciens and Clostridium celerecrescens. Moreover, the dominant bacteria of Yaks were also mainly belonged to phyla firmicutes and bacteroidetes, and the known dominant bacteria were including Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Schwartzia succinivorans and Clostridiales bacterium, most of them are common rumen bacteria. In addition, the dominant bacteria in Yunnan Yellow Cattle were belonged to phyla firmicutes, bacteroidetes and Actinobacteria, and the known dominant bacteria containing Prevotella sp., Staphylococci lentus, Staphylococcus xylosus and Corynebacterium casei. Present study first detected Staphylococcus lentus and Staphylococcus xylosus in the rumen of cattle.     

Formation of Lysine from α,ε-Diaminopimelic Acid and Negligible Synthesis of Lysine from Some Other Precursors by Rumen Ciliate Protozoa

The possibility of lysine formation from α,ε-diaminopimelate (DAP), acetate, aspartate or α-aminoadipate (AAA) in rumen ciliates was examined. DAP-1,7-¹⁴C added to the medium was decarboxylated and converted to radioactive lysine in great amounts and radioactive pipecolate in small amounts by rumen ciliates. Difference of the ability to form lysine from DAP between genus Entodinium and Diplodinium was not observed. With sodium acetate-U-¹⁴C, amino acids fraction of the supernatant fluid of the incubation medium and ciliates contained only 0.56 and 0.59% of the total radioactivity, respectively. In the case of l-aspartate-U-¹⁴C, 95.1% of the radioactivity of the supernatant fluid desalted and 62.2% of the radioactivity incorporated into ciliates (1.5% of the total radioactivity) remained as aspartate. Autoradiograms revealed the negligible spots of lysine in ciliates in both cases. AAA-6-¹⁴C remained almost unchanged, even after incubation with rumen ciliates.     

Postinoculation Protozoan Establishment and Association Patterns of Methanogenic Archaea in the Ovine Rumen

Association patterns between archaea and rumen protozoa were evaluated by analyzing archaeal 16S rRNA gene clone libraries from ovine rumen inoculated with different protozoa. Five protozoan inoculation treatments, fauna free (negative control), holotrich and cellulolytic protozoa, Isotricha and Dasytricha spp., Entodinium spp., and total fauna (type A) were tested. We used denaturing gradient gel electrophoresis, quantitative PCR, and phylogenetic analysis to evaluate the impact of the protozoan inoculants on the respective archaeal communities. Protozoan 18S ribosomal DNA clone libraries were also evaluated to monitor the protozoal population that was established by the inoculation. Phylogenetic analysis suggested that archaeal clones associated with the fauna-free, the Entodinium, and the type A inoculations clustered primarily with uncultured phylotypes. Polyplastron multivesiculatum was the predominant protozoan strain established by the holotrich and cellulolytic protozoan treatment, and this resulted predominantly in archaeal clones affiliated with uncultured and cultured methanogenic phylotypes (Methanosphaera stadtmanae, Methanobrevibacter ruminantium, and Methanobacterium bryantii). Furthermore, the Isotricha and Dasytricha inoculation treatment resulted primarily in archaeal clones affiliated with Methanobrevibacter smithii. This report provides the first assessment of the influence of protozoa on archaea within the rumen microbial community and provides evidence to suggest that different archaeal phylotypes associate with specific groups of protozoa. The observed patterns may be linked to the evolution of commensal and symbiotic relationships between archaea and protozoa in the ovine rumen environment. This report further underscores the prevalence and potential importance of a rather large group of uncultivated archaea in the ovine rumen, probably unrelated to known methanogens and undocumented in the bovine rumen.     

Molecular Phylogeny of the Family Ophryscolecidae (Ciliophora,Litostomatea) Inferred from 18S rDNA Sequences

The 18S rDNA was used to infer oral ciliature patterns of evolution within the family Ophryoscolecidae, with the addition of five new sequences of ciliates from the genus Ostracodinium. Our data confirmed the monophyly of the subfamilies Entodiniinae and Ophryoscolecinae, but more analysis would be required for the definition of the status of the subfamily Diplodiniinae. The oral infraciliature patterns reflect evolutionary divergence in the family Ophryscolecidae, observing monophyly on Entodinium‐type, Diplodinium‐type, Ostracodinium‐type, Epidinium‐type, and Ophryoscolex‐type. The ancestral infraciliature of Entodinium‐type cannot be proven, however, the position of Entodinium‐type showed closer of Diplodinium‐type than Ophryoscolex‐type, corroborating previous studies using morphological characters. The observed inconsistencies reflect the need to increase the number of 18S rDNA sequences to family Ophryoscolecidae and investigate the evolution of this group using other molecular markers.     

Development and Validation of a Real-Time PCR Method To Quantify Rumen Protozoa and Examination of Variability between Entodinium Populations in Sheep Offered a Hay-Based Diet

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR () was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R² = 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10⁰ and 10⁶ entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.     

Culture of the Rumen Holotrich Ciliate Dasytricha ruminantium Schuberg

The successful cultivation of the anaerobic ciliate Dasytricha ruminantium is described. The cultures were established in a salts medium containing 30% clarified rumen fluid. Sucrose and extract of rumen holotrich protozoa were fed once daily for 2 to 4 hr, and Dasytricha was then transferred to medium free from these nutrients. Rumen fluid was essential. Omission of protozoal extract resulted in gradual death of the ciliates. Bovine serum satisfactorily substituted for the protozoal extract, but various rumen bacteria, extract of rumen bacteria, and extracts of plant materials could not. There was a positive correlation between formation of methane in the cultures and growth of the ciliates. It is possible that methane bacteria were ingested, but it is not excluded that survival of both dasytrichs and the methanogenic bacteria depended on a low redox potential of the medium.     

Phylogenetic Analyses Support Validity of Genus Eodinium (Ciliophora,Entodiniomorphida, Ophryoscolecidae)

The validity of genus Eodinium has been historically disputed due to morphological similarities with Diplodinium (absence of skeletal plates as well as adoral and dorsal ciliary zones at the same body level). To address this issue, the 18S rDNA of four Eodinium posterovesiculatum morphotypes and four Diplodinium anisacanthum morphotypes were sequenced and phylogenetically analyzed. The different inference methods suggest the existence of a last common ancestor of Eodinium and Ostracodinium that is not shared with Diplodinium, strongly supporting the validity of genus Eodinium. Since skeletal plates are present in all members of genus Ostracodinium, the most parsimonious is a secondary loss of skeletal plates in E. posterovesiculatum. This work represents a breakthrough in the taxonomy and phylogeny of the family Ophryoscolecidae indicating that the skeletal plates may not reflect evolutionary divergence within this group of ciliates as traditionally proposed.     

Effect of dietary sodium bicarbonate supplementation on fermentation characteristics and ciliate protozoal population in rumen of lambs

Effects of sodium bicarbonate (NaHCO₃) inclusion on rumen fermentation characteristics and ciliate protozoal population in high concentrate-fed lambs were studied. Twenty-four weaner (90 days old) Malpura lambs divided into four equal groups (G1, G2, G3 and G4) were fed basal (25:75 roughage and concentrate) diet (G1) or basal diet supplemented with 0.75% (G2), 1.50% (G3) and 2.25% (G4) sodium bicarbonate for 90 days. Daily dry matter (DM) intake and digestibility of organic matter (OM), crude protein (CP), gross energy and plane of nutrition were similar in all the groups while cellulose digestibility was higher in G4 than in G2 and G1. Ruminal pH increased (P<0.05) with increasing levels of dietary sodium bicarbonate. Concentrations of total volatile fatty acids (TVFA), total nitrogen and trichloroacetic acid-precipitable nitrogen (TCA-ppt.-N) were higher while ammonia nitrogen was lower in the rumen fluid of G4 and G3 than in G2 and G1. The number of total protozoa, Isotricha, Dasytricha, large and small spirotrichs were higher (P<0.01) in the rumen of G4 and G3 than in G2 and G1. Total live weight gain and average daily gain were also higher in lambs supplemented with sodium bicarbonate. It is concluded that sodium bicarbonate inclusion at the rate of 1.50% of total ration increased cellulose digestibility, ciliate protozoal number, ruminal pH and total nitrogen concentration resulting in improved growth of lambs maintained on high concentrate diet.     

Rumen methanogen and protozoal communities of Tibetan sheep and Gansu Alpine Finewool sheep grazing on the Qinghai–Tibetan Plateau,China

Background

Tibetan sheep (TS) and Gansu Alpine Finewool sheep (GS) are both important plateau sheep raised and fed on the harsh Qinghai–Tibetan Plateau, China. Rumen methanogen and protozoal communities of plateau sheep are affected by their hosts and living environments, and play important roles in ruminant nutrition and greenhouse gas production. However, the characteristics, differences, and associations of these communities remain largely uncharacterized.

Results

The rumen methanogen and protozoal communities of plateau sheep were investigated by 16S/18S rRNA gene clone libraries. The predominant methanogen order in both sheep species was Methanobacteriales followed by Methanomassiliicoccales, which is consistent with those seen in global ruminants. However, the most dominant species was Methanobrevibacter millerae rather than Methanobrevibacter gottschalkii seen in most ruminants. Compared with GS and other ruminants, TS have more exclusive operational taxonomic units and a lower proportion (64.5%) of Methanobrevibacter. The protozoa were divided into Entodiniomorphida and Vestibuliferida, including nine genera and 15 species. The proportion of holotrich protozoa was much lower (1.1%) in TS than ordinary sheep. The most predominant genus was Entodinium (70.0%) in TS and Enoploplastron (48.8%) in GS, while the most common species was Entodinium furca monolobum (43.9%) and Enoploplastron triloricatum (45.0%) in TS and GS, respectively; Entodinium longinucleatum (22.8%) was only observed in TS. LIBSHUFF analysis indicated that the methanogen communities of TS were significantly different from those of GS, but no significant differences were found in protozoal communities.

Conclusion

Plateau sheep have coevolved with unique rumen methanogen and protozoal communities to adapt to harsh plateau environments. Moreover, the host appears to have a greater influence on rumen methanogen communities than on rumen protozoal communities. The observed associations of methanogens and protozoa, together with the findings of previous studies on methane emissions from ruminant livestock, revealed that the lower proportion of Methanobrevibacter and holotrich protozoa may be responsible for the lower methane emission of TS. These findings facilitate our understanding of the rumen microbial ecosystem in plateau sheep, and could help the development of new strategies to manipulate rumen microbes to improve productivity and reduce the emission of greenhouse gases.

     

Rumen Ciliates of Musk-Oxen (Ovibos moschatus Z.) from the Canadian Arctic1

Samples of rumen contents were obtained from 13 musk-oxen living on Banks Island, N.W.T., Canada. Total protozoan numbers ranged from 38.5 to 124.6 times 10⁴ per ml of rumen contents with a mean generic distribution of 52.4%Entodinium, 45.9%Diplodinium, and 1.7%Epidinium. A total of 18 protozoan species were found, six of which have not previously been observed in this host, i.e. D. (Diplodinium) costatum, D. (Ostracodinium) gracile, D. (Ostracodinium) trivesiculatum. D. (Eudiplodinium) medium, Epidinium quadricaudatum, and Epidinium parvicaudatum. Two new species of protozoa are described, Entodinium ovibos n. sp. and Diplodinium (Eudiplodinium) banksi n. sp.     

Phylogenetic Study of Methanogens Associated with Rumen Ciliates

The phylogeny of methanogenic archaea associated with ciliate protozoa in a sheep rumen was investigated. Ruminal ciliate protozoa were exhaustively washed and mixtures of genomic DNA extracted. Archaea-specific nested PCR amplification was conducted with the ciliate genomic mixture. The resultant small subunit (16S) ribosomal RNA gene (ssu rDNA) was cloned into Escherichia coli JM 109. Many methanogens were still observed on and/or in ciliate cells by fluorescent microscopy even after exhaustive washing with buffer. Partial sequences of ssu rDNA close to Methanobrevibacter smithii were dominant in the retrieved sequences. RFLP analyses on the retrieved sequences revealed the absence of Methanobrevibacter ruminantium in the protozoal preparation. The association of Methanobrevibacter spp. with ruminal ciliate protozoa was demonstrated by the isolation of archaeal ssu rDNA phylogenetically close to that of M. smithii. Received: 16 February 1999 / Accepted: 20 April 1999     

Diplodinium (Ostracodinium) minorum sp. n., Ciliate from the Rumen of Domestic Sheep*

SYNOPSIS. Diplodinium (Ostracodinium) minorum sp. n., observed in rumen contents from several domestic sheep, resembles Diplodinium (O.) ypsilon and Diplodinium (O.) magnum. It differs from the latter 2 species primarily in the size and proportions of the body, size of the rectum and anus, and in the presence of a short right caudal lobe in many individuals. In the samples studied, D. (O.) minorum constituted from 0.06 to 0.67% of the total rumen ciliate population.     

Rumen ciliates from Tanzanian short horn zebu cattle,Bos taurus indicus,and the infraciliature of Entodinium palmare n.sp. and Enoploplastron stokyi ()

Rumen ciliates of ten Tanzanian short horn zebu cattle were examined. A total of 15 genera and 46 species were identified, including a new Entodinium species. The ciliate density was 22.2×10⁴ ml^?1. The number of species per host and the diversity index showed high values, 33.8 and 2.80, respectively. Rumen ciliates had a low percentage composition of the genus Entodinium (7.0–25.0%) and a slightly higher percentage composition of the genera, Eudiplodinium (19.3%), Diplodinium (14.1%), and Ostracodinium (13.1%). Entodinium palmare n.sp., Eudiplodinium kenyensis, and Enoploplastron stokyi were found in all cattle examined. The former two species have been found only in African zebu cattle. Entodinium palmare n. sp. has a characteristic right surface of the body like the “palm of a hand” because of the concave part on the postero-dorsal part of the body, and has the same pattern of infraciliary bands as in other Entodinium species. Enoploplastron stokyi has a characteristic pattern of infraciliary bands analogous to those of Epidinium ecaudatum and Ostracodinium mammosum; with the right side of the adoral polybrachykinety gradually tapering and a slender short vestibular polybrachykinety.     

Rumen Ciliate Fauna of Alaskan Moose (Alces americana), Musk-Ox (Ovibos moschatus) and Dall Mountain Sheep (Ovis dalli)*

Total numbers, generic distribution and percentage species distribution were determined for the ciliate protozoa in rumen contents obtained from Alaskan moose (Alces americana), musk-ox (Ovibos moschatus) and Dall mountain sheep (Ovis dalli). The musk-ox has a fauna somewhat similar to that previously observed in reindeer and caribou. In contrast, only protozoa in the genus Entodinium were observed in moose, while Dall mountain sheep have a fauna unique among Alaskan ruminants studied to date. Other than Entodinium exiguum which was common to all animals, only 2 additional species of Entodinium, observed in the moose and musk-ox, occurred in more than one animal species. Four new species of protozoa are described, Entodinium dalli sp.n., Entodinium constrictum sp.n. and Polyplastron alaskum sp.n. from the Dall mountain sheep and Entodinium alces sp.n. from moose.     

Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.     

Comparison of nisin and monensin effects on ciliate and selected bacterial populations in artificial rumen

The effect of daily supplementation of nisin (2 mg/L), monensin (5.88 mg/L) and nisin and monensin (2 + 5.88 mg/L) on ovine ruminal ciliates and bacteria was investigated using the artificial rumen RUSITEC. Major groups in RUSITEC were Entodinium spp. and Dasytricha ruminantium. The supplementation of nisin significantly increased the population of both major ciliate groups. The supplementation of monensin significantly decreased the population of both groups. The combined effect of nisin and monensin was similar to the effect of monensin. Monensin had strong antiprotozoic effects in contrast to the stimulatory effects of nisin. D. ruminantium followed by Entodinium spp. appeared more resistant to tested compounds than other rumen ciliates. Tested additives did not significantly influence the presence and growth of amylolytic streptococci and enterococci but nisin showed a tendency to decreasing the concentration of Escherichia coli and lactobacilli.     

Impact of High-Concentrate Feeding and Low Ruminal pH on Methanogens and Protozoa in the Rumen of Dairy Cows

Non-lactating dairy cattle were transitioned to a high-concentrate diet to investigate the effect of ruminal pH suppression, commonly found in dairy cattle, on the density, diversity, and community structure of rumen methanogens, as well as the density of rumen protozoa. Four ruminally cannulated cows were fed a hay diet and transitioned to a 65% grain and 35% hay diet. The cattle were maintained on an high-concentrate diet for 3 weeks before the transition back to an hay diet, which was fed for an additional 3 weeks. Rumen fluid and solids and fecal samples were obtained prior to feeding during weeks 0 (hay), 1, and 3 (high-concentrate), and 4 and 6 (hay). Subacute ruminal acidosis was induced during week 1. During week 3 of the experiment, there was a significant increase in the number of protozoa present in the rumen fluid (P = 0.049) and rumen solids (P = 0.004), and a significant reduction in protozoa in the rumen fluid in week 6 (P = 0.003). No significant effect of diet on density of rumen methanogens was found in any samples, as determined by real-time PCR. Clone libraries were constructed for weeks 0, 3, and 6, and the methanogen diversity of week 3 was found to differ from week 6. Week 3 was also found to have a significantly altered methanogen community structure, compared to the other weeks. Twenty-two unique 16S rRNA phylotypes were identified, three of which were found only during high-concentrate feeding, three were found during both phases of hay feeding, and seven were found in all three clone libraries. The genus Methanobrevibacter comprised 99% of the clones present. The rumen fluid at weeks 0, 3, and 6 of all the animals was found to contain a type A protozoal population. Ultimately, high-concentrate feeding did not significantly affect the density of rumen methanogens, but did alter methanogen diversity and community structure, as well as protozoal density within the rumen of nonlactating dairy cattle. Therefore, it may be necessary to monitor the rumen methanogen and protozoal communities of dairy cattle susceptible to depressed pH when methane abatement strategies are being investigated.