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Effects of disodium fumarate on ruminal fermentation and microbial communities in sheep fed on high-forage diets

  • Y. W. Zhou (a1), C. S. McSweeney (a2), J. K. Wang (a1) and J. X. Liu (a1)


This study was conducted to investigate effects of disodium fumarate (DF) on fermentation characteristics and microbial populations in the rumen of Hu sheep fed on high-forage diets. Two complementary feeding trials were conducted. In Trial 1, six Hu sheep fitted with ruminal cannulae were randomly allocated to a 2 × 2 cross-over design involving dietary treatments of either 0 or 20 g DF daily. Total DNA was extracted from the fluid- and solid-associated rumen microbes, respectively. Numbers of 16S rDNA gene copies associated with rumen methanogens and bacteria, and 18S rDNA gene copies associated with rumen protozoa and fungi were measured using real-time PCR, and expressed as proportion of total rumen bacteria 16S rDNA. Ruminal pH decreased in the DF group compared with the control (P < 0.05). Total volatile fatty acids increased (P < 0.001), but butyrate decreased (P < 0.01). Addition of DF inhibited the growth of methanogens, protozoa, fungi and Ruminococcus flavefaciens in fluid samples. Both Ruminococcus albus and Butyrivibrio fibrisolvens populations increased (P < 0.001) in particle-associated samples. Trial 2 was conducted to investigate the adaptive response of rumen microbes to DF. Three cannulated sheep were fed on basal diet for 2 weeks and continuously for 4 weeks with supplementation of DF at a level of 20 g/day. Ruminal samples were collected every week to analyze fermentation parameters and microbial populations. No effects of DF were observed on pH, acetate and butyrate (P > 0.05). Populations of methanogens and R. flavefaciens decreased in the fluid samples (P < 0.001), whereas addition of DF stimulated the population of solid-associated Fibrobacter succinogenes. Population of R. albus increased in the 2nd to 4th week in fluid-associated samples and was threefold higher in the 4th week than control week in solid samples. Analysis of denaturing gradient gel electrophoresis fingerprints revealed that there were significant changes in rumen microbiota after adding DF. Ten of 15 clone sequences from cut-out bands appearing in both the 2nd and the 4th week were 94% to 100% similar to Prevotella-like bacteria, and four sequences showed 95% to 98% similarity to Selenomonas dianae. Another 15 sequences were obtained from bands, which appeared in the 4th week only. Thirteen of these 15 sequences showed 95% to 99% similarity to Clostridium sp., and the other two showed 95% and 100% similarity to Ruminococcus sp. In summary, the microorganisms positively responding to DF addition were the cellulolytic bacteria, R. albus, F. succinogenes and B. fibrisolvens as well as proteolytic bacteria, B. fibrisolvens, P. ruminicola and Clostridium sp.


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Arakaki, LC, Gaggiotti, MC, Cannillia, ML, Valtorta, S, Gallardo, MR, Conti, RG, Gregoret, F, Quaino, O, Kudo, H, Takenaka, A 2005. Evaluation of soybean silage in dairy cows under grazing conditions in Argentina: effects on rumen microorganisms. Proceedings of Japanese Society for Rumen Metabolism and Physiology 16, 87.
Asanuma, N, Hino, T 2000. Activity and properties of fumarate reductase in ruminal bacteria. The Journal of General and Applied Microbiology 46, 119125.
Asanuma, N, Iwamoto, M, Hino, T 1999a. The production of formate, a substrate for methanogenesis, from compounds related with the glyoxylate cycle by mixed ruminal microbes. Animal Science Journal 70, 6773.
Asanuma, N, Iwamoto, M, Hino, T 1999b. Effect of the addition of fumarate on methane production by ruminal microorganisms in vitro. Journal of Dairy Science 82, 780787.
Carro, MD, Ranilla, MJ 2003. Influence of different concentrations of disodium fumarate on methane production and fermentation of concentrate feeds by rumen micro-organisms in vitro. British Journal of Nutrition 90, 617623.
Castillo, C, Benedito, JL, Méndez, J, Pereira, V, López-Alonso, M, Miranda, M, Hernández, J 2004. Organic acids as a substitute for monensin in diets for beef cattle. Animal Feed Science and Technology 115, 101116.
Chen, XL, Wang, JK, Wu, YM, Liu, JX 2007. Effect of form of nitrogen on populations of fibre-associated ruminal microbes in pre-treated rice straw in vitro. Journal of Animal and Feed Sciences 16, 95100.
Chen, XL, Wang, JK, Wu, YM, Liu, JX 2008. Effects of chemical treatments of rice straw on rumen fermentation characteristics, fibrolytic enzyme activities and populations of liquid- and solid-associated ruminal microbes in vitro. Animal Feed Science and Technology 141, 114.
Denman, SE, McSweeney, CS 2006. Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiology Ecology 58, 572582.
Denman, SE, Tomkins, NW, McSweeney, CS 2007. Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS Microbiology Ecology 62, 313322.
Finlay, BJ, Esteban, G, Clarke, KJ, Williams, AG, Embley, T, Hirt, RP 1994. Some rumen ciliates have endosymbiotic methanogens. FEMS Microbiology Letters 117, 157161.
Forsberg, CW, Cheng, KJ, White, BA 1997. Polysaccharide degradation in the rumen and large intestine. In Gastrointestinal microbiology (ed. RI Mackie and BA White), pp. 319379. Chapman and Hall, New York, USA.
García-Martínez, R, Ranilla, MJ, Tejido, ML, Carro, MD 2005. Effects of disodium fumarate on in vitro rumen microbial growth, methane production and fermentation of diets differing in their forage : concentrate ratio. British Journal of Nutrition 94, 7177.
Hattori, K, Matsui, H 2008. Diversity of fumarate reducing bacteria in the bovine rumen revealed by culture dependent and independent approaches. Anaerobe 14, 8793.
Henderson, C 1980. The influence of extracellular hydrogen on the metabolism of Bacteroides ruminicola, Anaerovibrio lipolytica and Selenomonas ruminantium. Journal of General Microbiology 119, 485491.
Hillman, K, Lloyd, D, Williams, AG 1988. Interactions between the methanogen Methanosarcina barkeri and rumen holotrich ciliate protozoa. Letters in Applied Microbiology 7, 4953.
Hu, WL, Liu, JX, Ye, JA, Wu, YM, Guo, YQ 2005. Effect of tea saponin on rumen fermentation in vitro. Animal Feed Science and Technology 120, 333339.
Hungate, RE 1967. Hydrogen as an intermediate in the rumen fermentation. Archives of Microbiology 59, 158164.
Ji, YT, Qu, CQ, Cao, BY 2007. Optimized method of DNA silver staining in polyacylamide gels electrophoresis. Electrophoresis 28, 11731175.
Joblin, KN, Naylor, GE, Williams, AG 1990. Effect of methanobrevibacter smithii on xylanolytic activity of anaerobic ruminal fungi. Applied and Environmental Microbiology 56, 22872295.
Kocherginskaya, SA, Cann, IKO, Mackie, RI 2005. Denaturing gradient gel electrophoresis. In Methods in gut microbial ecology for ruminants (ed. HPS Makkar and CS McSweeney), pp. 119128. Springer, Dordrecht, the Netherlands.
Koike, S, Kobayashi, Y 2001. Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FEMS Microbiology Letters 204, 361366.
Krumholz, LR, Forsberg, CW, Veira, DM 1983. Association of methanogenic bacteria with rumen protozoa. Canadian Journal of Microbiology 29, 676680.
Makkar, HPS, McSweeney, CS 2005. Methods in gut microbial ecology for ruminants. Springer, Dordrecht, the Netherlands.
Marvin-Sikkema, FD, Richardson, AJ, Stewart, CS, Gottschal, JC, Prins, RA 1990. Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi. Applied and Environmental Microbiology 56, 37933797.
McAllister, TA, Newbold, CJ 2008. Redirecting rumen fermentation to reduce methanogenesis. Australian Journal of Experimental Agriculture 48, 713.
Ministry of Agriculture of China 2004. Feeding standard of meat-producing sheep and goats (NY/T 816-2004). China Agricultural Press, Beijing, China.
Morgavi, DP, Forano, E, Martin, C, Newbold, CJ 2010. Microbial ecosystem and methanogenesis in ruminants. Animal 4, 10241036.
Muyzer, G, de Waal, EC, Uitterlinden, AG 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695700.
Pavlostathis, SG, Miller, TL, Wolin, MJ 1990. Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii. Applied Microbiology and Biotechnology 33, 109116.
Russell, JB, Wallace, RJ 1997. Energy-yielding and energy-consuming reactions. In The rumen microbial ecosystem, 2nd edition (ed. PN Hobson and CS Stewart), pp. 246282. Blackie Academic and Professional, London, UK.
Searle, LP 1984. The berthelot or indophenol reaction and its use in the analytical chemistry of nitrogen: a review. Analyst 109, 549568.
SPSS 2006. SPSS Base 13.0 for Windows user's guide. SPSS Inc., Chicago, IL.
Stewart, CS, Flint, HJ, Bryant, MP 1988. The rumen bacteria. In The rumen microbial ecosystem, 1st edition (ed. PN Hobson), pp. 2175. Elsevier Applied Science, New York, USA.
Stumm, CK, Gijzen, HJ, Vogels, GD 1982. Association of methanogenic bacteria with ovine rumen ciliates. British Journal of Nutrition 47, 9599.
Ungerfeld, EM, Kohn, RA 2006. The role of thermodynamics in the control of ruminal fermentation. In Ruminant physiology. Digestion, metabolism and impact of nutrition on gene expression, immunology and stress (ed. K Sejrsen, T Hvelplund and MO Nielsen), pp. 5585. Wageningen Academic Publishers, Wageningen, the Netherlands.
Ungerfeld, EM, Kohn, RA, Wallace, RJ, Newbold, CJ 2007. A meta-analysis of fumarate effects on methane production in ruminal batch cultures. Journal of Animal Science 85, 25562563.
Ushida, K, Jouany, J 1996. Methane production associated with rumen-ciliated protozoa and its effect on protozoan activity. Letters in Applied Microbiology 23, 129132.
Wallace, RJ, Onodera, R, Cotta, MA 1997. Metabolism of nitrogen-containing compounds. In The rumen microbial ecosystem, 2nd edition (ed. PN Hobson and CS Stewart), pp. 283328. Blackie Academic & Professiional, London, UK.
Williams, AG, Coleman, GS 1997. The rumen protozoa. In The rumen microbial ecosystem, 2nd edition (ed. PN Hobson and CS Stewart), pp. 73139. Blackie Academic & Professiional, London, UK.
Williams, AG, Withers, SE, Joblin, KN 1994. The effect of cocultivation with hydrogen-consuming bacteria on xylanolysis by Ruminococcus flavefaciens. Current Microbiology 29, 133138.
Wolin, MJ 1974. Metabolic interactions among intestinal microorganisms. American Journal of Clinical Nutrition 27, 13201328.
Wolin, MJ 1979. The rumen fermentation: a model for microbial interactions in anaerobic ecosystems. In Advances in microbial ecology (ed. M Alexander), Vol. 3 pp. 4977. Plenum Press, New York.
Wolin, M, Miller, T, Stewart, C 1997. Microbe–microbe interactions. In The rumen microbial ecosystem (ed. PN Hobson and CS Stewart), pp. 467491. Blackie Academic & Professional, London, UK.
Wood, TA, Wallace, RJ, Rowe, A, Price, J, Yáñez-Ruiz, DR, Murray, P, Newbold, CJ 2009. Encapsulated fumaric acid as a feed ingredient to decrease ruminal methane emissions. Animal Feed Science and Technology 152, 6271.
Yu, CW, Chen, YS, Cheng, YH, Cheng, YS, Yang, CMJ, Chang, CT 2010. Effects of fumarate on ruminal ammonia accumulation and fiber digestion in vitro and nutrient utilization in dairy does. Journal of Dairy Science 93, 701710.


Effects of disodium fumarate on ruminal fermentation and microbial communities in sheep fed on high-forage diets

  • Y. W. Zhou (a1), C. S. McSweeney (a2), J. K. Wang (a1) and J. X. Liu (a1)


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