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Table 1 Techniques used to define the association between rumen protozoa and methanogens in 14 references

From: Rumen methanogens and mitigation of methane emission by anti-methanogenic compounds and substances

Techniques Description Methanogen population Host ciliate Animals & Diet & Sampling Reference
Culture-based enumeration MPN numbers of methanogens per ciliate cells were measured after each time points after feeding Maximum number of methanogens are detected 1 h after feeding (103 to 104 MPN/cell) Polyplastron
Entodinium spp.
- Animals : Sheep
- Diet : Mixed diet
- Sampling : 0, 1, 2 and 3 h after feeding
(in vitro culture)
Culture-based isolation & repeated washing + RFLP 1. Isolation of culturable methanogens from ciliate fraction on selective media
2. Retrieve the 16S rRNA sequences from washed ciliate fraction
isolates MB-9 - > a Mbb. ruminantium
Mbb. smithii related sequences were dominant
Ciliates fraction - Wethers
- Mixed diet (twice a day)
- 1 h after morning feeding
[27, 29]
Postinoculation of various protozoal fauna in defaunated sheep and notify the different archaeal phylotypes depends on the specific groups of rumen ciliates Predominant associated archaea species;
Isotrichidae—Mbb. smithii
P. multivesiculatumMbb. bryantii, Mbb.
stadtmanae, and Mbb. ruminantium
Holotrichs—uncultured archaea
4 different types of fauna - Wethers
- Corn silage + SBM
- Before morning feeding
Microbial population shift after long-term defaunation (methanogenic archaea & fibrolytic bacteria) Abundance of methanogens ↑, w/no difference on diversity in the absence of protozoa Entodiniomorphs (97%)
Holotrichs (3%)
- Wethers (in vivo)
- Mixed diet (once daily)
- Just before feeding
Short & long-term defaunation effect on the association between rumen protozoa and methanogens Defaunated and faunated samples from the liquid phase were placed in an independent cluster (DGGE) 3.8 × 106/ml ciliate cells
(95% Entodiniomorphs, 1.2%
Isotricha and 2.9%
- Wethers
- Mixed diet (twice a day)
- 3 h after morning feeding
Protozoal fractions
(w/nylon meshes of 80, 60, 45, 35, 20 and 5 μm pore diameters) were made by size fractionation.
No difference of methanogens abundance in- and out-side of ciliate cells.
Holotrichs has different methanogen community compared to the total protozoal fraction.
T-RFLP—Clear differences between PAM & free-living methanogens.
Low similarity among each protozoal fractions
Holotrich protozoa & total protozoa fraction - Sheep
- Mixed diet (twice a day)
- Before morning feeding
Repeated washing Washed protozoa fraction from monofaunated rumen fluid was used for DNA extraction.
Phylogenetic analysis was done with sequences.
The associated methanogens are highly correlated with the species in the rumen fluid.
All sequences showed high similarity to the family Methanobacteriaceae Isotricha prostoma
Eudiplodinium maggii
- Sheep’s rumen (monofaunated) [28]
Repeated washing + qPCR mcrA & 16S rRNA gene was amplified from washed protozoal fraction.
Construction of clone library with amplicons for phylogenetic analysis
qPCR for quantification of each methanogenic group (Mbb, bMm, cRCC)
Methanomicrobium spp. was mostly found in free living environment
Mbb (free living-18, PAM-34%)
Mm (free living-25, PAM-17%)
RCC (free living-58, PAM-48%)
Forage - > high grain diet (RCC↓, Mbb↑)
Ciliates fraction - Heifers
- Forage fed - > highconcentrate diet/d
- 1 h prior to feeding
Single cell isolation Extracellular microbes were removed by antibiotics treatment.
16S rRNA gene sequences were amplified from the isolated single cells of each protozoal species and sequenced.
Methanobrevibacter sp. was the most abundant genus among three ciliates.
Minor detection of Methanomicrobium sp. and RCC group were found.
Isotricha intestinalis
Ophryoscolex purkynjei.
- Goat’s rumen (in vitro) [30]
Single cell isolation Methanogen population distributed to each protozoal species analyzed by single cell isolation followed by sequencing of SSU rRNA genes Retrieved 20 novel sequences had low identity to the known sequences in the databases.
Methanimicrococcus baltticola & Mm. mobile were the most related known species among the protozoa species.
Ophryoscolex caudatus
Metadinium medium
Entodinium furca
Diplodinium dentatum
- Sheep, Cow and Goat's rumen + Sheep’s rumen (in vitro) [31]
Single cell isolation + DGGE 16S rRNA gene was amplified from the isolated
Entodinium caudatum cells and applied to DGGE
Only one DGGE band was shown from isolated single cell.
The sequence only found from isolated Ento cell not in the total DNA.
Entodinium caudatum (Long-term in vitro cultured) - Sheep’s rumen (in vitro) [176]
FISH probing FISH was applied to detect prokaryotes colonized in various protozoal species D. ruminantium (archaea (−))
Isotricha spp. (37.5% archaea (+))
P. multivesticulatum (archaea (−))
Epidinium spp. (16.3% archaea (+))
Eu. maggii (8% archaea (+))
Entodinium spp. (42.8% archaea (+))
5 different types of fauna - Sheep
- Hay (ad libitum) + pelleted concentrate/d
- Before feeding
FISH probing FISH was applied to detect and quantify the associated methanogens in Entodinium spp.. Methanogens including Mbb. thaueri, Mbb.millerae and Mbb. smithii, and members of Mm. and Methanospaera spp. were generally the predominant colonizers of protozoa.
Entodinium spp. were colonized by similar methanogenic populations regardless of the forage fed.
Entodinium spp. Cattle
- Alfalfa hay or triticale straw
- After feeding (1–2 h)
  1. aMbb = Methanobrevibacter
  2. bMm = Methanomicrobium
  3. cRCC = rumen cluster C