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RESEARCH ARTICLE

Changes in ruminal microbiota due to rumen content processing and incubation in single-flow continuous-culture fermenters

E. C. Soto A , D. R. Yáñez-Ruiz A B , G. Cantalapiedra-Hijar A , A. Vivas A and E. Molina-Alcaide A
+ Author Affiliations
- Author Affiliations

A Estación Experimental del Zaidín (Consejo Superior de Investigaciones Científicas), Professor Albareda, 1, 18008 Granada, Spain.

B Corresponding author. Email: david.yanez@eez.csic.es

Animal Production Science 52(9) 813-822 https://doi.org/10.1071/AN11312
Submitted: 14 November 2011  Accepted: 28 February 2012   Published: 3 July 2012

Abstract

The aim of this study was to investigate the impact of rumen content manipulation and its incubation in an in vitro system on the abundance of some microbial groups and the bacterial diversity of goat rumens. Animals and single-flow continuous-culture fermenters were fed diets differing in forage to concentrate ratio (70 : 30; LC and 30 : 70; HC). Rumen contents were sampled after animals’ adaptation to the experimental diets, processed for inoculum preparation and inoculated into fermenters. Fermenter contents were sampled 1 and 7 days after inoculation. Total bacteria, Fibrobacter succinogenes, fungi and methanogen abundances were lower in the fermenter than in goat rumens, but no differences were found for Ruminococcus flavefaciens. The abundances of all these microorganisms were similar at 1 and 7 days of rumen content incubation in fermenters. Bacterial species richness did not change due to rumen content processing or the in vitro incubation. Shannon–Wiener index and Pielou evenness were lower in the fermenter than in rumen only when the enzyme HaeIII was used in terminal-restriction fragment length polymorphism analysis. Non-metric multidimensional scaling analysis, both in denaturing gradient gel electrophoresis and terminal-restriction fragment length polymorphism, showed a segregation of in vivo and in vitro samples, but no trends of grouping for fermenter samples was observed. The HC diet promoted higher abundance of total bacteria than LC in rumen but not in fermenters. Diet only had an effect on bacterial diversity when the enzyme HaeIII was considered. Rumen content processing and incubation in fermenters caused an important decline of the studied ruminal microbial groups although bacterial community structure and diversity did not significantly change.


References

AOAC (2005) ‘Official methods of analysis.’ 18th edn. (Association of Official Analytical Chemists: Gaithersburg, MD)

Cantalapiedra-Hijar G, Yáñez-Ruiz DR, Martín-García AI, Molina-Alcaide E (2009) Effects of forage: concentrate ratio and forage type on apparent digestibility, ruminal fermentation, and microbial growth in goats. Journal of Animal Science 87, 622–631.
Effects of forage: concentrate ratio and forage type on apparent digestibility, ruminal fermentation, and microbial growth in goats.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFGns78%3D&md5=5f2f6468379d72297e1479fec5ee0ea2CAS |

Cantalapiedra-Hijar G, Yáñez-Ruiz DR, Newbold CJ, Molina-Alcaide E (2011) The effect of the feed-to-buffer ratio on bacterial diversity and ruminal fermentation in single-flow continuous-culture fermenters. Journal of Dairy Science 87, 622–631.

Castillejos L, Calsamiglia S, Ferret A, Losa R (2007) Effects of dose and adaptation time of a specific blend of essential oil compounds on rumen fermentation. Animal Feed Science and Technology 132, 186–201.
Effects of dose and adaptation time of a specific blend of essential oil compounds on rumen fermentation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xht1eqtrvF&md5=5070e720b8aa9c2a3f2ceec7f21a6683CAS |

Cheng KJ, McAllister TA, Costerton JW (1995) Biofilms of the ruminant digestive tract. In ‘Microbial biofilms’. (Eds HM Lappin-Scott, JW Costerton) pp. 221–232. (Cambridge University Press: Cambridge, UK)

Cho SJ, Cho KM, Shin EC, Lim WJ, Hong SY, Choi BR, Kang JM, Lee SM, Kim YH, Hoon K, Yun HD (2006) 16S rDNA analysis of bacterial diversity in three fractions of cow rumen. Journal of Microbiology and Biotechnology 16, 92–101.

Clarke KR (1993) Non-parametric multivariate analyses of changes in community structure. Australian Journal of Ecology 18, 117–143.
Non-parametric multivariate analyses of changes in community structure.Crossref | GoogleScholarGoogle Scholar |

Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM (2009) The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Research 37, D141–D145.
The Ribosomal Database Project: improved alignments and new tools for rRNA analysis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhsFejtLbF&md5=ae08a60538ddf594be63df3b76c55b4eCAS |

Crawford RJ, Hoover WH, Knowlton PH (1980) Effects of solids and liquid flows on fermentation in continuous cultures. I. Dry matter and fiber digestion, VFA production and protozoa numbers. Journal of Animal Science 51, 975–985.

Czerkawski JW (1969) Methane production in ruminants and its significance. World Review of Nutrition and Dietetics 11, 240–282.

Denman SE, McSweeney CS (2005) Quantitative (real-time) PCR. In ‘Methods in gut microbial ecology for ruminants’. (Eds HPS Makkar, CS McSweeney) pp. 105–115. (Springer: Dordrecht, The Netherlands)

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, 572–582.
Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XhtlSmtLrO&md5=ffe33154701ed73361b607036e6dd0d8CAS |

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, 313–322.
Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsValtr7E&md5=17fa47552cbe554bfd16eaa9bb87d17eCAS |

Engebretson JJ, Moyer CL (2003) Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism. Applied and Environmental Microbiology 69, 4823–4829.
Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXmsFWrtLk%3D&md5=48f2df0bd6c96b51e5cb7caf3723ec27CAS |

Gill M, Smith P, Wilkinson JM (2010) Mitigating climate change: the role of domestic livestock. Animal 4, 323–333.
Mitigating climate change: the role of domestic livestock.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC38vptFaisA%3D%3D&md5=c374796a696eb88111e98d9abec84b44CAS |

Gizzi G, Zanchi R, Sciaraffia F (1998) Comparison of microbiological and fermentation parameters obtained with an improved rumen in vitro technique with those obtained in vivo. Animal Feed Science and Technology 73, 291–305.
Comparison of microbiological and fermentation parameters obtained with an improved rumen in vitro technique with those obtained in vivo.Crossref | GoogleScholarGoogle Scholar |

Gordon GL, Phillips MW (1989) Degradation and utilization of cellulose and straw by three different anaerobic fungi from the ovine rumen. Applied and Environmental Microbiology 55, 1703–1710.

Hongoh Y, Yuzawa H, Ohkuma M, Kudo T (2003) Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment. FEMS Microbiology Letters 221, 299–304.
Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXjtlSlu7c%3D&md5=46c7229666ac0560bb7b0dd412ad0fd1CAS |

Kajikawa H, Jin H, Terada F, Suga T (2003) Operation and characteristics of newly improved and marketable artificial rumen (Rusitec). Memoirs of the National Institute of Livestock and Grassland Science 2, 1–21. [Japan]

Kent AD, Smith DJ, Benson BJ, Triplett EW (2003) Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities. Applied and Environmental Microbiology 69, 6768–6776.
Web-based phylogenetic assignment tool for analysis of terminal restriction fragment length polymorphism profiles of microbial communities.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXptVyktLc%3D&md5=3a3f14318aafa3a8c0dc52c8c5793b82CAS |

Khafipour E, Li S, Plaizier JC, Krause DO (2009) Rumen microbiome composition determined using two nutritional models of subacute ruminal acidosis. Applied and Environmental Microbiology 75, 7115–7124.
Rumen microbiome composition determined using two nutritional models of subacute ruminal acidosis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFSit7vN&md5=a54e72be153b3d71b268c098d162ce0eCAS |

Kobayashi Y, Shinkai T, Koike S (2008) Ecological and physiological characterization shows that Fibrobacter succinogenes is important in rumen fiber digestion – Review. Folia Microbiologica 53, 195–200.
Ecological and physiological characterization shows that Fibrobacter succinogenes is important in rumen fiber digestion – Review.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXovF2hs70%3D&md5=7c840793ab043561eba3d6ba2516fb29CAS |

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, 361–366.
Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXos1Kiur8%3D&md5=aee04921377cc5e75225b1b1435be5fbCAS |

Koike S, Yoshitani S, Kobayashi Y, Tanaka K (2003) Phylogenetic analysis of fiber-associated rumen bacterial community and PCR detection of uncultured bacteria. FEMS Microbiology Letters 229, 23–30.
Phylogenetic analysis of fiber-associated rumen bacterial community and PCR detection of uncultured bacteria.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXptlOqs78%3D&md5=554ba663b25f1858c42263e17b83e458CAS |

Kong Y, Teather R, Forster R (2010) Composition, spatial distribution, and diversity of the bacterial communities in the rumen of cows fed different forages. FEMS Microbiology Ecology 74, 612–622.
Composition, spatial distribution, and diversity of the bacterial communities in the rumen of cows fed different forages.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhsFKmsrvK&md5=a7229dd8715d737b0fdc5d8705426748CAS |

Kruskal JB, Wish M (1978) ‘Multidimensional scaling.’ (Sage Publications: Beverly Hills, CA)

Latham MJ, Brooker BE, Pettipher GL, Harris PJ (1978) Adhesion of Bacteroides succinogenes in pure culture and in the presence of Ruminococcus flavefaciens to cell walls in leaves of perennial ryegrass (Lolium perenne). Applied and Environmental Microbiology 35, 1166–1173.

Legendre P, Legendre L (1998) ‘Numerical ecology.’ 2nd edn. (Elsevier: Amsterdam, The Netherlands)

Li M, Gong J, Cottrill M, Yu H, de Lange C, Burton J, Topp E (2003) Evaluation of QIAamp DNA Stool Mini Kit for ecological studies of gut microbiota. Journal of Microbiological Methods 54, 13–20.

Loesche WJ (1969) Oxygen sensitivity of various anaerobic bacteria. Applied Microbiology 18, 723–727.

Mackie RI, Gilchrist FMC, Robberts AM, Hannah PE, Schwartz HM (1978) Microbiological and chemical changes in the rumen during the stepwise adaptation of sheep to high concentrate diets. The Journal of Agricultural Science 90, 241–254.
Microbiological and chemical changes in the rumen during the stepwise adaptation of sheep to high concentrate diets.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1cXkvV2gsb0%3D&md5=1ecee4fbadc7986656f5ea2ff29a7ef1CAS |

Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, Arai H, Tanimoto I, Nishimura F, Takashiba S (2003) Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunology and Medical Microbiology 39, 81–86.
Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXnvFWitrg%3D&md5=0df4f718fa5ffb641aab7f28156a6200CAS |

Mansfield HR, Endres MI, Stern MD (1995) Comparison of microbial fermentation in the rumen of dairy cows and dual flow continuous culture. Animal Feed Science and Technology 55, 47–66.
Comparison of microbial fermentation in the rumen of dairy cows and dual flow continuous culture.Crossref | GoogleScholarGoogle Scholar |

Martínez ME, Ranilla MJ, Tejido ML, Ramos S, Carro MD (2010a) Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. I. Digestibility, fermentation parameters, and microbial growth. Journal of Dairy Science 93, 3684–3698.
Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. I. Digestibility, fermentation parameters, and microbial growth.Crossref | GoogleScholarGoogle Scholar |

Martínez ME, Ranilla MJ, Tejido ML, Saro C, Carro MD (2010b) Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. II. Protozoa population and diversity of bacterial communities. Journal of Dairy Science 93, 3699–3712.
Comparison of fermentation of diets of variable composition and microbial populations in the rumen of sheep and Rusitec fermenters. II. Protozoa population and diversity of bacterial communities.Crossref | GoogleScholarGoogle Scholar |

McDougall EI (1948) Studies on ruminant saliva. 1. The composition and output of sheep’s saliva. Biochemical Journal 43, 99–109.

Michalet-Doreau B, Fernandez I, Peyron C, Millet L, Fonty G (2001) Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents. Reproduction, Nutrition, Development 41, 187–194.
Fibrolytic activities and cellulolytic bacterial community structure in the solid and liquid phases of rumen contents.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3MznvVChuw%3D%3D&md5=0ca55376b220a7d97a728bb70174a800CAS |

Miettinen H, Setälä J (1989) Design and development of a continuous culture system for studying rumen fermentation. Journal of Agricultural Science in Finland 61, 463–473.

Moeseneder MM, Arrieta JM, Muyzer G, Winter C, Herndl GJ (1999) Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 65, 3518–3525.

Molina-Alcaide E, Pascual MR, Cantalapiedra-Hijar G, Morales-García EY, Martín-García AI (2009) Effects of concentrate replacement by feed blocks on ruminal fermentation and microbial growth in goats and single-flow continuous-culture fermenters. Journal of Animal Science 87, 1321–1333.
Effects of concentrate replacement by feed blocks on ruminal fermentation and microbial growth in goats and single-flow continuous-culture fermenters.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXjsFKis70%3D&md5=28b2db57e390ea40be3475d8936728f7CAS |

Moumen A, Yáñez-Ruiz DR, Martín-García AI, Molina-Alcaide E (2009) Protozoa evolution in single-flow continuous-culture fermenters and Rusitec fermenters fed high-forage diets. Options Méditerranéenes 85, 303–308.

Mountfort DO (1987) The rumen anaerobic fungi. FEMS Microbiology Letters 46, 401–408.
The rumen anaerobic fungi.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL2sXmtFCms7c%3D&md5=b66c383e4502fd5fae297787f1dde5cbCAS |

Muetzel S, Lawrence P, Hoffmann EM, Becker K (2009) Evaluation of a stratified continuous rumen incubation system. Animal Feed Science and Technology 151, 32–43.
Evaluation of a stratified continuous rumen incubation system.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXltlOisL8%3D&md5=19476434150d4f92db8f02df098a995cCAS |

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, 695–700.

Pielou EC (1966) The measurement of diversity in different types of biological collections. Journal of Theoretical Biology 13, 131–144.
The measurement of diversity in different types of biological collections.Crossref | GoogleScholarGoogle Scholar |

Prevot S, Senaud J, Bohatier J, Prensier G (1994) Variation in the composition of the ruminal bacterial microflora during the adaptation phase in an artificial fermentor (Rusitec). Zoological Science 11, 871–878.

Prieto C, Aguilera JF, Lara L, Fonollá J (1990) Protein and energy requirements for maintenance of indigenous Granadina goats. The British Journal of Nutrition 63, 155–163.
Protein and energy requirements for maintenance of indigenous Granadina goats.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK3c3ktFygtg%3D%3D&md5=0a146cf654c629d493105ebba01b3f81CAS |

Sadet S, Martin C, Meunier B, Morgavi DP (2007) PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium. Animal 1, 939–944.
PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtFGqtL%2FK&md5=dca115f04305b2ad113f12b479b5ecbbCAS |

Sadet-Bourgeteau S, Martin C, Morgavi DP (2010) Bacterial diversity dynamics in rumen epithelium of wethers fed forage and mixed concentrate forage diets. Veterinary Microbiology 146, 98–104.
Bacterial diversity dynamics in rumen epithelium of wethers fed forage and mixed concentrate forage diets.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BC3cbjtVKkug%3D%3D&md5=19dfb53b43121949a68ef5db55aa1807CAS |

Shannon CE, Weaver W (1949) ‘The mathematical theory of information.’ (University of Illinois Press: Urbana, IL)

Shinkai T, Kobayashi Y (2007) Localization of ruminal cellulolytic bacteria on plant fibrous materials as determined by fluorescence in situ hybridization and real-time PCR. Applied and Environmental Microbiology 73, 1646–1652.
Localization of ruminal cellulolytic bacteria on plant fibrous materials as determined by fluorescence in situ hybridization and real-time PCR.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXjsVSktLw%3D&md5=b5026d4ccfb70fec7508a3fffa610a02CAS |

Shyu C, Soule T, Bent SJ, Foster JA, Forney LJ (2007) MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes. Microbial Ecology 53, 562–570.
MiCA: a web-based tool for the analysis of microbial communities based on terminal-restriction fragment length polymorphisms of 16S and 18S rRNA genes.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXltlSlu7w%3D&md5=fb03745d785eeb57f1874cb011b747ebCAS |

Sørensen T (1948) A method of establishing groups of equal amplitude in plant sociology based on similarity of species content and its application to analyses of the vegetation on Danish Commons. Biologiske Skrifter 5, 1–34.

Van den Abbeele P, Grootaert C, Marzorati M, Possemiers S, Verstraete W, Gerard P, Rabot S, Bruneau A, El Aidy S, Zoetendal E, Kleerebezem M, Smidt H, Van de Wiele T (2010) Microbial community development in a dynamic gut model is reproducible, colon-region specific and selects for Bacteroidetes and Clostridium Cluster IX. Applied and Environmental Microbiology 76, 5237–5246.
Microbial community development in a dynamic gut model is reproducible, colon-region specific and selects for Bacteroidetes and Clostridium Cluster IX.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtFWnurjM&md5=769f49f968ac66181cc78bb1c96a5b1dCAS |

Van Soest PJ, Robertson JB, Lewis B (1991) Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 3583–3597.
Methods for dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK38%2FnvVCltA%3D%3D&md5=2648256a724beb4754b35e474c24f1b0CAS |

Wiener N (1948) ‘Cybernetics or communication and control in the animal and the machine.’ (John Wiley and Sons: New York)

Williams AG (1986) Rumen holotrich ciliate protozoa. Microbiology and Molecular Biology Reviews 50, 25–49.

Yu Z, Yu M, Morrison M (2006) Improved serial analysis of V1 ribosomal sequence tags (SARST V1) provides a rapid, comprehensive, sequence based characterization of bacterial diversity and community composition. Environmental Microbiology 8, 603–611.
Improved serial analysis of V1 ribosomal sequence tags (SARST V1) provides a rapid, comprehensive, sequence based characterization of bacterial diversity and community composition.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xkt1Kqurg%3D&md5=ea0e4c28cf9dd698fd7f2fa0e9f259c6CAS |

Ziemer CJ, Sharp R, Stern MD, Cotta MA, Whitehead TR, Stahl DA (2000) Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes. Environmental Microbiology 2, 632–643.
Comparison of microbial populations in model and natural rumens using 16S ribosomal RNA-targeted probes.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD3M7otVWltA%3D%3D&md5=00651d1b12b71904ac89ffa805cf46e4CAS |