Effects of Rumen Protozoa of Brahman Heifers and Nitrate on Fermentation and In vitro Methane Production

  • Nguyen, S.H. (School of Environmental and Rural Sciences, University of New England) ;
  • Li, L. (School of Environmental and Rural Sciences, University of New England) ;
  • Hegarty, R.S. (School of Environmental and Rural Sciences, University of New England)
  • Received : 2015.07.30
  • Accepted : 2015.12.22
  • Published : 2016.06.01


Two experiments were conducted assessing the effects of presence or absence of rumen protozoa and dietary nitrate addition on rumen fermentation characteristics and in vitro methane production in Brahman heifers. The first experiment assessed changes in rumen fermentation pattern and in vitro methane production post-refaunation and the second experiment investigated whether addition of nitrate to the incubation would give rise to methane mitigation additional to that contributed by defaunation. Ten Brahman heifers were progressively adapted to a diet containing 4.5% coconut oil distillate for 18 d and then all heifers were defaunated using sodium 1-(2-sulfonatooxyethoxy) dodecane (Empicol). After 15 d, the heifers were given a second dose of Empicol. Fifteen days after the second dosing, all heifers were allocated to defaunated or refaunated groups by stratified randomisation, and the experiment commenced (d 0). On d 0, an oral dose of rumen fluid collected from unrelated faunated cattle was used to inoculate 5 heifers and form a refaunated group so that the effects of re-establishment of protozoa on fermentation characteristics could be investigated. Samples of rumen fluid collected from each animal using oesophageal intubation before feeding on d 0, 7, 14, and 21 were incubated for in vitro methane production. On d 35, 2% nitrate (as $NaNO_3$) was included in in vitro incubations to test for additivity of nitrate and absence of protozoa effects on fermentation and methane production. It was concluded that increasing protozoal numbers were associated with increased methane production in refaunated heifers 7, 14, and 21 d after refaunation. Methane production rate was significantly higher from refaunated heifers than from defaunated heifers 35 d after refaunation. Concentration and proportions of major volatile fatty acids, however, were not affected by protozoal treatments. There is scope for further reducing methane output through combining defaunation and dietary nitrate as the addition of nitrate in the defaunated heifers resulted in 86% reduction in methane production in vitro.


Defaunation;Refaunation;Nitrate;Fermentation and Methane Production


  1. Allison, M. J. and C. A. Reddy. 1984. Adaptations of gastrointestinal bacteria in response to changes in dietary oxalate and nitrate. In: Third International Symposium on Microbial Ecology (Eds. M. J. Klug and C. A. Reddy). Washington, DC, USA. Am. Soc. Microbiol. pp. 248-256.
  2. Bird, S. H., R. S. Hegarty, and R. Woodgate. 2008. Persistence of defaunation effects on digestion and methane production in ewes. Aust. J. Exp. Agric. 48:152-155.
  3. Bird, S. H. and M. N. Light. 2013. A protocol for the removal of protozoa (defaunation) from the rumen of cattle. In: Proceeding of the Recent Advances in Animal Nutrition in Australia (Ed. P. Cronje). University of New England Publishing Unit, Armidale, Australia. pp. 1-2.
  4. Dehority, B. A. 1984. Evaluation of subsampling and fixation procedure used for counting rumen protozoa. Appl. Environ. Microbiol. 48:182-185.
  5. Eugene, M., H. Archimede, and D. Sauvant. 2004. Quantitative meta-analysis on the effects of defaunation of the rumen on growth, intake and digestion in ruminants. Livest. Prod. Sci. 85:81-97.
  6. Finlay, B. J., G. Esteban, K. J. Clarke, A. G. Williams, T. M. Embley, and R. P. Hirt. 1994. Some rumen ciliates have endosymbiotic methanogens. FEMS Microbiol. Lett. 117:157-161.
  7. Guo, W. S., D. M. Schaefer, X. X. Guo, L. P. Ren, and Q. X. Meng. 2009. Use of nitrate-nitrogen as a sole dietary nitrogen source to inhibit ruminal methanogenesis and to improve microbial nitrogen synthesis in vitro. Asian Australas. J. Anim. Sci. 22:542-549.
  8. Hegarty, R. S., S. H. Bird, B. A. Vanselow, and R. Woodgate. 2008. Effects of the absence of protozoa from birth or from weaning on the growth and methane production of lambs. Br. J. Nutr. 100:1220-1227.
  9. Jouany, J. P., D. I. Demeyer, and J. Grain. 1988. Effect of defaunating the rumen. Anim. Feed Sci. Technol. 21:229-265.
  10. Leng, R. A. and T. R. Preston. 2010. Further considerations of the potential of nitrate as a high affinity electron acceptor to lower enteric methane production in ruminants. Livest. Res. Rural Dev. 22:221.
  11. Lin, M., D. M. Schaefer, W. S. Guo, L. P. Ren, and Q. X. Meng. 2011. Comparisons of in vitro nitrate reduction, methanogenesis, and fermentation acid profile among rumen bacterial, protozoal and fungal fractions. Asian Australas. J. Anim. Sci. 24:471-478.
  12. Morgavi, D. P., E. Forano, C. Martin, and C. J. Newbold. 2010. Microbial ecosystem and methanogenesis in ruminants. Animal 4:1024-1036.
  13. Morgavi, D. P., J. P. Jouany, and C. Martin. 2008. Changes in methane emission and rumen fermentation parameters induced by refaunation in sheep. Aust. J. Exp. Agric. 48:69-72.
  14. Morgavi, D. P., C. Martin, J. P. Jouany, and M. Ranilla. 2012. Rumen protozoa and methanogenesis: not a simple causeeffect relationship. Br. J. Nutr. 107:388-397.
  15. Newbold, C. J., B. Lassalas, and J. P. Jouany. 1995. The importance of methanogensis associated with ciliate protozoa in ruminal methane production in vitro. Lett. Appl. Microbiol. 21:230-234.
  16. Newbold, C. J., G. de la Fuente, A. Belanche, E. Ramos-Morales, and N. McEwan. 2015. The role of ciliate protozoa in the rumen. Front. Microbiol. 6:Article 1313.
  17. Nolan, J. V., R. S. Hegarty, J. Hegarty, I. R. Godwin, and R. Woodgate. 2010. Effects of dietary nitrate on fermentation, methane production and digesta kinetics in sheep. Anim. Prod. Sci. 50:801-806.
  18. Qin, W. Z., C. Y. Li, J. K. Kim, J. G. Ju, and M. K. Song. 2012. Effects of defaunation on fermentation characteristics and methane production by rumen microbes in vitro when incubated with starchy feed sources. Asian Australas. J. Anim. Sci. 25:1381-1388.
  19. Ranilla, M. J., J. P. Jouany, and D. P. Morgavi. 2007. Methane production and substrate degradation by rumen microbial communities containing single protozoal species in vitro. Lett. Appl. Microbiol. 45:675-680.
  20. Santra, A. and S. A. Karim. 2002. Influence of ciliate protozoa on biochemical changes and hydrolytic enzyme profile in the rumen ecosystem. J. Appl. Microbiol. 92:801-811.
  21. Santra, A., S. A. Karim, and O. H. Chaturvedi. 2007. Rumen enzyme profile and fermentation characteristics in sheep as affected by treatment with sodium lauryl sulfate as defaunating agent and presence of ciliate protozoa. Small Rumin. Res. 67:126-137.
  22. Soliva, C. R. and H. D. Hess. 2007. Measuring methane emission of ruminants by in vitro and in vivo. In: Measuring Methane Production from Ruminants (Eds. P. S. M. Harinder and E. V. Philip). Springer, Netherlands. pp. 15-31.
  23. Stumm, C. K., H. J. Gijzen, and G. D. Vogels. 1982. Association of methanogenic bacteria with ovine rumen ciliates. Br. J. Nutr. 47:95-99.
  24. van Zijderveld, S. M., W. J. J. Gerrits, J. A. Apajalahti, J. R. Newbold, J. Dijkstra, R. A. Leng, and H. B. Perdok. 2010. Nitrate and sulfate: Effective alternative hydrogen sinks for mitigation of ruminal methane production in sheep. J. Dairy. Sci. 93:5856-5866.
  25. van Zijderveld, S. M., W. J. J. Gerrits, J. Dijkstra, J. R. Newbold, R. B. A. Hulshof, and H. B. Perdok. 2011. Persistency of methane mitigation by dietary nitrate supplementation in dairy cows. J. Dairy Sci. 94:4028-4038.