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Amplified Ribosomal DNA Restriction Analysis Method to Assess Rumen Microbial Diversity of Ruminant

Authors: A. Natsir, M. Nadir, S. Syahrir, A. Mujnisa, N. Purnomo, A. R. Egan, B. J. Leury


Rumen degradation characteristic of feedstuff is one of the prominent factors affecting microbial population in rumen of animal. High rumen degradation rate of faba bean protein may lead to inconstant rumen conditions that could have a prominent impact on rumen microbial diversity. Amplified Ribosomal DNA Restriction Analysis (ARDRA) is utilized to monitor diversity of rumen microbes on sheep fed low quality forage supplemented by faba beans. Four mature merino sheep with existing rumen cannula were used in this study according to 4 x 4 Latin square design. The results of study indicated that there were 37 different ARDRA types identified out of 136 clones examined. Among those clones, five main clone types existed across the treatments with different percentages. In conclusion, the ARDRA method is potential to be used as a routine tool to assess the temporary changes in the rumen community as a result of different feeding strategies.

Keywords: Microbial diversity, Ruminants, clones, ARDRA method, ribotypes

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[1] K. Ntushelo, “Identifying bacteria and studying bacterial diversity using the 16s ribosomal RNA-gene-based sequencing techniques: A review,” Afr. J. Microbiol. Res., Vol. 7, no. 49, pp. 5533-5540, 2013.
[2] D. O. Krause and J. B. Russel,” How many ruminal bacteria are there?” J. Dairy Sci., Vol 79, pp. 1467-1475, 1996.
[3] J. L. Firkins and Z. Yu,” Chracterisation and quantification of the microbial population of the rumen,” in Digestion, Metabolism and Impact of Nutrition on Gene Expression, Immunology and Stress, K. Serjen et al Eds. the Netherelands: Wageningen Academic Publishers, 2008, pp. 19-54.
[4] S. Koike, J. Pan, Y. Kobayashi, and K. Tanaka,” Kinetics of in sacco fiber-attachment of representative ruminal cellulolytic bacteria monitored by competitive PCR,” J. Dairy Sci., Vol. 86, pp. 1429-1435, 2003.
[5] J.T. Sylvester, S. K. R. Karnati, Z. Yu, M. Morrison, and J. L. Firkins,” Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR,” J. Nutr., Vol. 134, pp. 3378-3384, 2004
[6] E. C. Shin, K. M. Cho, W. J. Lim, S. Y. Hong, C. L. An, K. M. Cho, Y. K. Kim, J. M. An, J. M. Kang, S. S. Lee, H. Kim, and H. D. Yun,” Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence,” Anaerobe,” Vol. 10, pp. 313- 319, 2004.
[7] W. Deng, M. Wanapat, S. Ma, J. Chen, D. Xi, T. He, Z. Yang, H. Mao,” Phylogeneic analysis of 16S rDNA sequences manifest rumen bacterial diversity in gayals (Bos frontalis) fed fresh bamboo leaves and twigs,” Asian-Aust. J. Anim. Sci., Vol. 20, no. 7, pp. 1057-1066, 2007.
[8] W. D. Hiorns, R.C. Hastings, I. M. Head, A. J. McCarthy, J. R. Saunders, R. W. Pickup, and G.H. Hall,” Amplification of 16s ribosomal RNA genes of autotrophic ammonia oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment,” Microbiology, vol. 141, pp. 2793-2800, 1995.
[9] F. B. Gich, E. Amer, J. B. Figueras, C. A. Abella, M. D. Balaguer, and M. Poch,” Assessment of microbial community structure by amplified ribosomal DNA restriction analysis (ARDRA),” Int. Microbiol., Vol. 3, pp. 103, 2000.
[10] M. Shah,” Amplified ribosomal DNA restriction analysis as a tool to characterize microbial community structure of activated sludge of common effluent treatment plant,” International Journal of Environmental Bioremediation & Biodegradation, Vol. 2, no.4, pp. 197-201, 2014.
[11] D. Błaszczyk, I. Bednarek, G. Machnik, D. Sypniewski, D. Sołtysik, T. Loch, and S. Gałka,” Amplified ribosomal DNA restriction analysis (ARDRA) as a screening method for normal and bulking activated sludge sample differentiation,” Polish J. of Environ. Stud, Vol. 20, no 1, pp. 29, 2011.
[12] D. S. Petterson and J. B. Mackintosh, The Chemical Composition and nutritive Value of Australian Grain Legumes. Canberra: Grain Research and Development Corporation, pp. 13-16, 1994.
[13] P. Yu, A. R. Egan, and B.J. Leury, “Protein evaluation of dry roasted whole faba beans (Vicia faba) and whole lupins seeds (Lupinus albus) by the new Duch protein evaluation system: the DVE/OEB systems,” Asia-Aust. J. Anim. Sci., Vol. 12, pp. 871-880, 1999.
[14] P. Yu, A. R. Egan, J. H. G. Holmes, and B. J. Leury,” Influence of dry roasting of whole faba beans (Vicia faba) on rumen degradation characteristics in dairy cows. II: starch,” Asian-Austr. J. Anim. Sci., Vol. 11, no. 5, pp. 503-509, 1998.
[15] P. Yu, J. O. Goelema, and S. Tamminga,” Using the DVE/OEB model to determine optimal conditions for pressure toasting on horse beans (Vicia faba) for the dairy feed industry,” Anim. Feed Sci. Technol., Vol. 86, pp. 165-176, 2000.
[16] M. A. Cotta and J. B. Russell,” Effect of peptides and amino acids on efficiency of rumen bacterial protein synthesis in continuous culture,” J. Dairy Sci., Vol. 65, pp. 226-234,1982.
[17] A. Natsir,” Purine derivative excretion and estimated of microbial protein supply on shep fed on different protein supplements,” JITV, Vol. 12, no. 3, pp. 183-188, 2007.
[18] A. Natsir, “Effects of different feeding frequency of faba beans on rumen degradation characteristics of oaten hay in the rumen of sheep,” Anim. Prod., Vol. 10, no.1, pp. 60, 2008.
[19] A. Natsir, A. R. Egan, B. J. Leury, M. Brandon, and C. Cunningham, “Dietary fiber attributes and effects of high grain feeding on the fibre degradation dynamics in dairy cows,” The Australian Journal of Dairy Technology, Vol. 56, no.2, pp. 160, 2001.
[20] A. Natsir, Fibre Utilization by Ruminants, Makassar, South Sulawesi: Masagena Press, 2012.
[21] R.G.D. Steel and J. H. Torrie, Principles and Procedures of Statistics. A Biometrical Approach. New York: McGraw-Hill International Book Company, 2nd Ed., 1981.
[22] L. Boonek, Y. J. Williams, B. J. Leury, and A. R. Egan,” microbial diversity in rumen ecosystem of lactating cows grazing different levels of highly digestible persian clover,” Anim. Prod. Aust., Vol. 24: 29-32, 2002.
[23] S. Kumar, K. Tamura, I. B. Jakobsen, and M. Nei,” MEGA2: Molecular Evolutionary genetics software, Bioinformatics, 2001.
[24] M. Nei and W.H. Li,” Mathematical model for studying genetic variation in terms of restriction endonuclease,” Proc. Natl. Acad. Sci. Vol. 76: 5269-5273. 1979.
[25] J. B. Russell and D.B. Wilson,” Why are ruminal cellulolytic bacteria unable to digest cellulose at low pH?” J. Dairy Sci., Vol. 79, pp. 1503-1509. 1996.
[26] S. Peters, S. Koschinsky, F. Schwieger, C. C. Tebbe,” Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes,” Appl. Environ. Microbiol., Vol. 66, no. 3, pp.930, 2000.
[27] J. Maukonen, J. Mättö, G. Wirtanen, L. Raaska, T. Mattila-Sandholm, M. Saarela” Methodologies for the characterization of microbes in industrial environments: A review,” J. Ind. Microbiol. Biotechnol., Vol. 3o, pp. 327, 2003.
[28] B. Thurston, K. A. Dawson, and H. J. Strobel,” Cellobiose versus glucose utilizationby ruminal bacterium Ruminococcus albus,” Applied and Environmental Microbiology, Vol. 59, no. 8, pp. 2631-2637, 1993.
[29] X. Hun, Y. Yang, H. Yan, X. Wang, L, Qu, and Y. Chen,” Rumen bacterial diversity of 80 to 110-day-old-goats using 16S rRNA sequencing,” Plos One, Vol. 10, no.2, pp. e0117811, 2015.
[30] H. Missa, A. Susilowati, and R. Setyaningsih,” Diversity and phylogenetic relationshipof cellulytic bacteria from the feces of bali cattle in South Central Timor, East Nusa tenggara, Indonesia,” Biodiversitas, Vol. 17, no.2, pp. 614-619, 2016.
[31] M.P. Bryant,” Ruminococcus,” in Manual of Systematic Bacteriology, Vol. 2, P.H.A. Sneath, B. Williams, and B. Wilkins Eds., pp. 1093-1097, 1986.
[32] Y. Van der Linden, N.O. Van Gylswyk, and H.M. Schwartz,” Influence of supplementation of corn stover with corn grain on the fibrolytic bacteria in the rumen of sheep and their relation to the intake and digestion of fibre,” J. Anim. Sci., Vol. 59, pp. 772-783, 1984.
[33] J. B. Russell and D.B. Wilson,” Potential opportunities and problems for genetically altered rumen microorganisms,” J. Nutr., Vol. 118, pp. 271, 1988.
[34] J. B. Russell and D. B. Dombrowski,” Effect of pH on the efficiency of growth by pure cultures of rumen bacteria in continuous culture,” Appl. Environ. Microbiol., Vol. 39, pp. 604-610, 1980.
[35] N. O. Van Gylswyk,” Succiniclasticum ruminis, sp. nov., a ruminal bacterium converting succinate to propionate as the sole energy-yielding mechanism,” Int. J. Syst. Bacteriol., Vol. 45, no. 2, pp. 297-300, 1995.
[36] F. Martin-Laurent, L. Philippot, S. Hallet, R. Chaussod, J. C. Germon, G. Soulas, G. Catroux,” DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods,” Appl. Environ. Microbiol., Vol. 67, no. 5, pp. 2354, 2001.
[37] M. T. Suzuki and S. J. Giovannoni,” Bias caused by annealing in the amplification of mixtures of 16S rRNA genes by PCR,” Appl. Environ. Microbiol., Vol. 62, pp. 625-630, 1996.
[38] M. Cardinale, L. Brusetti, P. Quatrini, S. Borin, A. M. Puglia, A. Rizzi, E. Zanardini, C. Sorlini, C. Corselli, and D. Daffonchio,” Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities”, Appl. Environ. Microbiol, Vol. 70, pp. 6147–6156, 2004.