Standardization and Improved Protocols for Evaluation of Relative Abundance of Methanogens in Rumen of Dairy Cows

Author: Suresh C., Ramachandran M., Puvarajan B., Suresh R. and Vijayakumar P.

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Abstract

Methanogens are obligate anaerobes. Ruminal methanogens are still difficult to be cultured in laboratories, owing to their tough cell wall. The conventional methods of DNA extraction would not be feasible as they are found to be qualitative rather than quantitative. Hence, an improved methodology was attempted by quantitative real-time PCR (qRT-PCR) technique for determining the abundance of methanogen in the rumen of cattle. The relative abundance of all methanogens as well as key species such as Methanobrevibacter sp., and Methanosphaera stadtmanae were determined in the rumen liquor of cattle fed with 60:40 roughage and concentrate diet in the farm with standard managemental conditions in the Cauvery Delta Zone of Tamilnadu. DNA extraction was followed in three processing methods. Gradient PCR and RT-PCR has been carried out by use of published primers. Methanosphera standmanae was observed as the more abundant species than Methanobrevibacter sp. in this study. The parameters for standardisation of RT-PCR such as annealing temperature, melting curve can be used in future as standard to conduct are search protocol. The area and feeding system specific standardised protocols can be widely adopted in the rumen analytical studies to assess relative abundance of methanogens.

Keywords

Methanogens, RT-PCR, Gradient PCR, Methane, Methanosphera standmanae, Rumen

Conclusion

Methanosphaera standmanae was higher in this present study than Methanobrevibacter sp. Hence, sampling can be done in the left paralumbar fossa site for specific study on Methanosphaera standmanae. Otherwise, the sampling of rumen liquor can be made through stomach tube from cranial sac of rumen where initial active fermentation takes place for routine studies. This site will be more representative for all rumen microbes than the site at left paralumbar fossa. The primers used in the study can be effectively used owing to the prudentiality and estimation of relative abundance of methanogens in Jersey cross bred dairy cows of this region with the existing managemental conditions. In DNA extraction protocol, column purification should be carried out. The procedure without column purification will not be useful to extract DNA to observe the abundance and quantification of the methanogens. The selection and designing of novel primers for different area specific feeding practices will be useful in study of kinetics of the methanogens in rumen. Hence, the above mentioned improved protocols of this study can be followed for estimating the relative abundance of methanogens in the rumen in paddy straw based feeding practices adopted areas.

References

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How to cite this article

Suresh C., Ramachandran M., Puvarajan B., Suresh R. and Vijayakumar P. (2023). Standardization and Improved Protocols for Evaluation of Relative Abundance of Methanogens in Rumen of Dairy Cows. Biological Forum – An International Journal, 15(8): 144-150.