Skip to main content Accessibility help

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and in batch cultures of rumen microorganisms

  • I. MATEOS (a1), M. J. RANILLA (a1) (a2), C. SARO (a1) and M. D. CARRO (a3)


The objective of the current study was to assess how closely batch cultures (BC) of rumen microorganisms can mimic the dietary differences in fermentation characteristics found in the rumen, and to analyse changes in bacterial diversity over the in vitro incubation period. Four ruminally and duodenally cannulated sheep were fed four diets having forage : concentrate ratios (FCR) of 70 : 30 or 30 : 70, with either alfalfa hay or grass hay as forage. Rumen fluid from each sheep was used to inoculate BC containing the same diet fed to the donor sheep, and the main rumen fermentation parameters were determined after 24 h of incubation. There were differences between BC and sheep in the magnitude of most measured parameters, but BC detected differences among diets due to forage type similar to those found in sheep. In contrast, BC did not reproduce the dietary differences due to FCR found in sheep for pH, degradability of neutral detergent fibre and total volatile fatty acid (VFA) concentrations. There were differences between systems in the magnitude of most determined parameters and BC showed higher pH values and NH3–N concentrations, but lower fibre degradability and VFA and lactate concentrations compared with sheep. There were significant relationships between in vivo and in vitro values for molar proportions of acetate, propionate and butyrate, and the acetate : propionate ratio. The automated ribosomal intergenic spacer analysis (ARISA) of 16S ribosomal deoxyribonucleic acid showed that FCR had no effect on bacterial diversity either in the sheep rumen fluid used as inoculum (IN) or in BC samples. In contrast, bacterial diversity was greater with alfalfa hay diets than those with grass hay in the IN, but was unaffected by forage type in the BC. Similarity index between the bacterial communities in the inocula and those in the BC ranged from 67·2 to 74·7%, and was unaffected by diet characteristics. Bacterial diversity was lower in BC than in the inocula with 14 peaks out of a total of 181 detected in the ARISA electropherograms never appearing in BC samples, which suggests that incubation conditions in the BC may have caused a selection of some bacterial strains. However, each BC sample showed the highest similarity index with its corresponding rumen IN, which highlights the importance of using rumen fluid from donors fed a diet similar to that being incubated in BC when conducting in vitro experiments.


Corresponding author

*To whom all correspondence should be addressed. Email:


Hide All
Association of Official Analytical Chemists (1999). Official Methods of Analysis, 16th edn, 5th revision. Gaithersburg, MD: AOAC International.
Brown, V. E., Rymer, C., Agnew, R. E. & Givens, D. I. (2002). Relationship between in vitro gas production profiles of forages and in vivo rumen fermentation patterns in beef steers fed those forages. Animal Feed Science and Technology 98, 1324.
Bryant, M. P. (1973). Nutritional requirements of the predominant rumen cellulolytic bacteria. Federation Proceedings 32, 18091813.
Carro, M. D., Lebzien, P. & Rohr, K. (1992). Influence of yeast culture on the ‘in vitro’ fermentation (Rusitec) of diets containing variable portions of concentrates. Animal Feed Science and Technology 37, 209220.
Carro, M. D., Valdés, C., Ranilla, M. J. & González, J. S. (2000). Effect of forage to concentrate ratio in the diet on ruminal fermentation and digesta flow kinetics in sheep. Animal Science 70, 127134.
Czerkawski, J. W. & Breckenridge, G. (1977). Design and development of a long-term rumen simulation technique (Rusitec). British Journal of Nutrition 38, 371384.
Faichney, G. J. (1975). The use of markers to partition digestion within the gastro-intestinal tract of ruminants. In Digestion and Metabolism in the Ruminant (Eds Warner, A. C. I. & McDonald, I. W.), pp. 277291. Armidale, New South Wales, Australia: University of New England Publishing Unit.
Goering, H. K. & Van Soest, P. J. (1970). Forage Fiber Analysis (Apparatus, Reagents, Procedures and some Applications). Agricultural Handbook, no. 379. Washington DC, USA: Agricultural Research Services, USDA.
Mackie, R. I., Gilchrist, F. M. C., Robberts, A. M., Hannah, P. E. & Schwartz, H. M. (1978). Microbiological and chemical changes in the rumen during the stepwise adaptation of sheep to high concentrate diets. Journal of Agricultural Science, Cambridge 90, 241254.
Mateos, I., Ranilla, M. J., Tejido, M. L., Saro, C., Kamel, C. & Carro, M. D. (2013). The influence of diet type (dairy versus intensive fattening) on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production. Animal Production Science 53, 299307.
Mould, F. L., Kliem, K. E., Morgan, R. & Mauricio, R. M. (2005). In vitro microbial inoculum: a review of its function and properties. Animal Feed Science and Technology 123–124, 3150.
Prates, A., De Oliveira, J. A., Abecia, L. & Fondevila, M. (2010). Effects of preservation procedures of rumen inoculum on in vitro microbial diversity and fermentation. Animal Feed Science and Technology 155, 186193.
Ramos, S., Tejido, M. L., Martínez, M. E., Ranilla, M. J. & Carro, M. D. (2009 a). Microbial protein synthesis, ruminal digestion, microbial populations, and nitrogen balance in sheep fed diets varying in forage-to-concentrate ratio and type of forage. Journal of Animal Science 87, 29242934.
Ramos, S., Tejido, M. L., Ranilla, M. J., Martínez, M. E., Saro, C. & Carro, M. D. (2009 b). Influence of detachment procedure and diet on recovery of solid-associated bacteria from sheep ruminal digesta and representativeness of bacterial isolates as assessed by automated ribosomal intergenic spacer analysis-polymerase chain reaction. Journal of Dairy Science 92, 56595668.
Ranilla, M. J., López, S., Giráldez, F. J., Valdés, C. & Carro, M. D. (1998). Comparative digestibility and digesta flow kinetics in two breeds of sheep. Animal Science 66, 389396.
Russell, J. B. & Wilson, D. B. (1996). Why are ruminal cellulolytic bacteria unable to digest cellulose at low pH? Journal of Dairy Science 79, 15031509.
Rymer, C. & Givens, D. I. (2002). Relationships between patterns of rumen fermentation measured in sheep and in situ degradability and the in vitro gas production profile of the diet. Animal Feed Science and Technology 101, 3144.
Saro, C., Ranilla, M. J. & Carro, M. D. (2012). Postprandial changes of fiber-degrading microbes in the rumen of sheep fed diets varying in type of forage as monitored by real-time PCR and automated ribosomal intergenic spacer analysis. Journal of Animal Science 90, 44874494.
SAS (2012). SAS Institute Inc. SAS/STAT 12·1 User's Guide. Cary, NC: SAS Institute Inc.
Taylor, K. A. C. C. (1996). A simple colorimetric assay for muramic acid and lactic acid. Applied Biochemistry and Biotechnology 56, 4958.
Tejido, M. L., Ranilla, M. J., García-Martínez, R. & Carro, M. D. (2005). In vitro microbial growth and rumen fermentation of different diets as affected by the addition of disodium malate. Animal Science 81, 3138.
Van Soest, P. J., Robertson, J. B. & Lewis, B. A. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 35833597.
Warner, A. C. I. (1956). Criteria for establishing the validity of in vitro studies with rumen micro-organisms in so-called artificial rumen systems. Journal of General Microbiology 14, 733748.
Weatherburn, M. W. (1967). Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry 39, 971974.
Yu, Z. & Morrison, M. (2004). Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques 36, 808812.
Ziemer, C. J., Sharp, R., Stern, M. D., Cotta, M. A., Whitehead, T. R. & Stahl, D. A. (2000). Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes. Environmental Microbiology 2, 632643.

Comparison of fermentation characteristics and bacterial diversity in the rumen of sheep and in batch cultures of rumen microorganisms

  • I. MATEOS (a1), M. J. RANILLA (a1) (a2), C. SARO (a1) and M. D. CARRO (a3)


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed