Effects of Aspergillus Oryzae Culture and 2-Hydroxy-4-(Methylthio)-Butanoic Acid on In vitro Rumen Fermentation and Microbial Populations between Different Roughage Sources

  • Sun, H. (Institute of Dairy Science, College of Animal Sciences, Zhejiang University) ;
  • Wu, Y.M. (Institute of Dairy Science, College of Animal Sciences, Zhejiang University) ;
  • Wang, Y.M. (Novus International Research Center) ;
  • Liu, J.X. (Institute of Dairy Science, College of Animal Sciences, Zhejiang University) ;
  • Myung, K.H. (Department of Animal Science, Chonnam National University)
  • Received : 2013.11.22
  • Accepted : 2014.01.31
  • Published : 2014.09.01


An in vitro experiment was conducted to evaluate the effects of Aspergillus oryzae culture (AOC) and 2-hydroxy-4-(methylthio)-butanoic acid (HMB) on rumen fermentation and microbial populations between different roughage sources. Two roughage sources (Chinese wild rye [CWR] vs corn silage [CS]) were assigned in a $2{\times}3$ factorial arrangement with HMB (0 or 15 mg) and AOC (0, 3, or 6 mg). Gas production (GP), microbial protein (MCP) and total volatile fatty acid (VFA) were increased in response to addition of HMB and AOC (p<0.01) for the two roughages. The HMB and AOC showed inconsistent effects on ammonia-N with different substrates. For CWR, neither HMB nor AOC had significant effect on molar proportion of individual VFA. For CS, acetate was increased (p = 0.02) and butyrate was decreased (p<0.01) by adding HMB and AOC. Increase of propionate was only occurred with AOC (p<0.01). Populations of protozoa ($p{\leq}0.03$) and fungi ($p{\leq}0.02$) of CWR were differently influenced by HMB and AOC. Percentages of F. succinogenes, R. albus, and R. flavefaciens (p<0.01) increased when AOC was added to CWR. For CS, HMB decreased the protozoa population (p = 0.01) and increased the populations of F. succinogenes and R. albus ($p{\leq}0.03$). Populations of fungi, F. succinogenes (p = 0.02) and R. flavefacien (p = 0.03) were increased by adding AOC. The HMB${\times}$AOC interactions were noted in MCP, fungi and R. flavefacien for CWR and GP, ammonia-N, MCP, total VFA, propionate, acetate/propionate (A/P) and R. albus for CS. It is inferred that addition of HMB and AOC could influence rumen fermentation of forages by increasing the number of rumen microbes.


Aspergillus Oryzae;2-Hydroxy-4-(Methylthio)-Butanoic Acid;Chinese Wild Rye;Corn Silage;In vitro Rumen Fermentation;Microbial Population


Supported by : Novus International Inc., the China Agriculture Research System


  1. Di Francia, A., F. Masucci, G. De Rosa, M. L. Varricchio, and V. Proto. 2008. Effects of Aspergillus oryzae extract and a Saccharomyces cerevisiae fermentation product on intake, body weight gain and digestibility in buffalo calves. Anim. Feed Sci. Technol. 140:67-77.
  2. Frumholtz, P. P., C. J. Newbold, and R. J. Wallace. 1989. Influence of Aspergillus oryzae fermentation extract on the fermentation of a basal ration in the rumen simulation technique (Rusitec). J. Agric. Sci. (Camb.) 113:169-172.
  3. Hu, W. L., J. X. Liu, J. A. Ye, Y. M. Wu, and Y. Q. Guo. 2005. Effects of tea saponin on rumen fermentation in vitro. Anim. Feed Sci. Technol. 120:333-339.
  4. AOAC. 1997. Official Methods of Analysis, 16th ed. Association of Official Analytical Chemists, Arlington, VA, USA.
  5. Wang, Y. and T. A. McAllister. 2002. Rumen microbes, enzymes and feed digestion-A review. Asian Australas. J. Anim. Sci. 15:1659-1676.
  6. Whelan, S. J., F. J. Mulligan, J. J. Callan, B. Flynn, and K. M. Pierce. 2013. Effect of forage source and a supplementary methionine hydroxyl analogue on rumen fermentation parameters in lactating dairy cows offered a low crude protein diet. Anim. Feed Sci. Technol. 183:62-66.
  7. Wilson, K. R., C. S. Abney, J. T. Vasconcelos, M. Vazquez-Anon, J. P. McMeniman, and M. L. Galyean. 2008. Effects of 2-hydroxy-4-(methylthio)-butanoic acid on performance and carcass characteristics of finishing beef cattle and on fermentation in continuous culture. J. Anim. Sci. 86:1951-1962.
  8. Blake, W. L., M. D. Stern, and S. M. Hannah. 1986. Effect of supplementing methionine in various forms on bacterial degradation of methionine in continuous culture. Nutr. Rep. Int. 33:729-738.
  9. Boing, J. T. P. 1983. Enzyme production. In: Industrial Microbiology (Ed. G. Reed). 4th ed. AVI Publ. Co., Inc., Westport, CT, USA. pp. 685-689.
  10. Carton, J. S., D. O. Erickson, D. A. Carey, and D. L. Ulmer. 1993. Influence of Aspergillus oryzae fermentation extract on forage intake, site of digestion, in situ degradability, and duodenal amino acid flow in steers grazing cool-season pasture. J. Anim. Sci. 71:779-787.
  11. Chen, C. R., B. Yu, and P. W. S. Chiou. 2004. Roughage energy and degradability estimation with Aspergillus oryzae inclusion using $Daisy^{(R)}$ in vitro fermentation. Asian Australas. J. Anim. Sci. 17:53-62.
  12. Chen, X. L., J. K. Wang, Y. M. Wu, and J. X. Liu. 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. Anim. Feed Sci. Technol. 141:1-14.
  13. Chung, Y. H., H. G. Bateman, C. C. Williams, C. C. Stanley, D. T. Gantt, T. W. Braud, L. L. Southern, J. D. Ward, P. G. Hoyt, and G. A. Sod. 2006. Effects of methionine and lysine on fermentation in vitro and in vivo, nutrient flow to the intestine, and milk production. J. Dairy Sci. 89:1613-1620.
  14. Denman, S. E. and C. S. McSweeney. 2006. Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol. Ecol. 58:572-582.
  15. Denman, S. E., N. W. Tomkins, and C. S. McSweeney. 2007. Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane. FEMS Microbiol. Ecol. 62:313-322.
  16. National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. Edn. National Academy Press, Washington, DC, USA.
  17. Newbold, J. 1997. Proposed mechanisms for enzymes as modifiers of ruminal fermentation. In: Processing of the 8th Annual Ruminant Nutrition Symposium, Gainesville, Florida, USA. pp. 146-159.
  18. Noftsger, S. M., N. R. St-Pierre, S. K. R. Karnati, and J. L. Firkins. 2003. Effects of 2-hdyroxy-4-(methylthio) butanoic acid (HMB) on microbial growth in continuous culture. J. Dairy Sci. 86:2629-2636.
  19. Orskov, E. R. and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. (Camb.) 92:499-503.
  20. SAS. 1999. SAS User′s Guide: Statistics, Version 8.0. SAS Inst Inc, Cary, NC, USA.
  21. Schmidt, J. A., S. Albright, G. M. Calza, and R. E. Calza. 2004. Characterization of Aspergillus oryzae fermentation extract effects on the rumen fungus Neocallimastix frontalis EB 188. Part 2. Carbon source utilization and effects on zoospore production. Appl. Microbiol. Biotechnol. 63:431-437.
  22. Van Soest, P. J., J. B. Bobertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583-3597.
  23. Vazquez-Anon, M., T. Cassidy, P. McCullough, and G. A. Varga. 2001. Effects of Alimet on nutrient digestibility, bacterial protein synthesis, and ruminal disappearance during continuous culture. J. Dairy Sci. 84:159-166.
  24. Autrey, K. M., J. A. McCaskey, and J. A. Little. 1975. Cellulose digestibility of fibrous materials treated with Trichoderma viride cellulase. J. Dairy Sci. 58:67-71.
  25. Beharka, A. A. and T. G. Nagaraja. 1993. Effect of Aspergillus oryzae fermentation extract $(Amaferm^{(R)})$ on in vitro fiber degradation. J. Dairy Sci. 76:812-818.
  26. Jouany, J. P., F. Mathieu, J. Senaud, J. Bohatier, G. Bertin, and M. Mercier. 1998. Effect of Saccharomyces cerevisiae and Aspergillus oryzae on the digestion of nitrogen in the rumen of defaunated and refaunated sheep. Anim. Feed Sci. Technol. 75:1-13.
  27. Koike, S. and Y. Kobayashi. 2001. Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococccus flavefacies. FEMS Microbiol. Lett. 204:361-366.
  28. Lee, S. S., C. K. Choi, B. H. Ahn, Y. H. Moon, C. H. Kim, and J. K. Ha. 2004. In vitro stimulation of rumen microbial fermentation by a rumen anaerobic fungal culture. Anim. Feed Sci. Technol. 115:215-226.
  29. Mao, H. L., J. K. Wang, Y. Y. Zhou, and J. X. Liu. 2010. Effect of addition of tea saponin and soybean oil on methane production, fermentation and microbial population in the rumen of growing lambs. Livest. Sci. 129:56-62.
  30. Martin C., C. Mirande, D. P. Morgavi, E. Forano, E. Devillard, and P. Mosoni. 2013. Methionine analogues HMB and HMBi increase the abundance of cellulolytic bacterial representatives in the rumen of cattle with no direct effects on fibre degradation. Anim. Feed Sci. Technol. 182:16-24.
  31. Menke, K. H. and H. Steingass. 1988. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28:7-55.