Effect of Live Bacterial Cultures on In Vitro Digestibility and Ruminal Fermentation Parameters

Document Type : Ruminant Nutrition


1 گنبد کاووس

2 Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

3 Department of Animal Science, Ahar Faculty of Agriculture and Natural Resources, University of Tabriz, Tabriz, Iran


Introduction: Rumen microbial manipulation has been of interest to ruminant nutritionists in order to achieve improvement in the profitability and health of lactating cows. Concerns regarding the use of antibiotics and other growth stimulants in animal feed industry have caused attention to find other alternative agents to replace antibiotics. For these reasons, in ruminant, microbial cultures have been used to replace antibiotics to enhance milk production in dairy cattle and improve feed efficiency and daily gain in beef cattle. Rumen bacteria, that have ability to ferment carbohydrate, are primarily responsible for causing lactic acidosis in ruminant. Propionibacteria are natural inhabitants of the rumen that comprise 1.4 % of the ruminal microflora and produce propionic and acetic acid in the rumen. Therefore, propionibacteria have been used as a direct-fed microbial to prevent the risk of acidosis in feedlot cattle. Last studies have reported that combinations of propionic bacteria and lactobacilli resulted in increased average daily gain and improved feed efficiency in feedlot cattle. The purpose of this study were preparation of live bacterial culture in laboratory and investigate the effect of adding live bacterial culture on digestibility and ruminal fermentation parameters on in vitro condition.
Materials and Methods: Three in vitro experiments designed in order to determine the effects of bacteria strains supplementation on dry matter and organic matter digestibility, pH, VFA and ammonia nitrogen concentration. Bacterial pure strains were Propioni bacterium freudenreichii and Enterococcus faecium. Stock cultures of freeze-dried strains were individually inoculated into 5 mL Brain Heart infusion (BHI) and sodium lactate broth (SLB) respectively and incubated at 37 °C for 24 h under anaerobic conditions. By pour plating serial 10 fold dilutions (in sterile ringer’s solution) on demand, Rogosa, sharp agar and SLB agar plates were incubated anaerobically at 37 °C for 48h. 108 cfu /ml of culture were produced after 24 and 36 h of culturing. Rumen fluid was collected 3 h after morning feeding from three ruminally fistulated sheep with mean body weight of 45+2.5 kg. Buffer was prepared as proposed by Goering and Van Soest (1970). In an anaerobic condition, 50 ml of buffered rumen fluid (ratio of buffer to rumen fluid was 1:1), was dispensed into a 100 ml serum bottle containing 0.5 g DM of the experimental diet (four replicate) for each experimental incubation time (2, 4, 6, 12 and 24 h). The experimental treatments were: CON) control (basal diet without any additive), P) basal diet inoculated with 108 cfu Propionibacteria feriderinrichii, E) basal diet inoculated with 108 cfu Enterococcus faecium and P+E) basal diet inoculated with 108 cfu Propionibacteria fredrinrichii + Enterococcus faecium. Rumen fluid was collected before morning feeding from three ruminally fistulated sheep. In all experiments a diet based on concentrate were used with the ratio 90: 10 of concentrate to forage. In the second experiment 1 mL sucrose (10% w/w) injected to the diet and in third experiment pH adjusted to 5.5 at the beginning of experiment. After 2, 4, 6, 12 and 24 h of the incubation, the bottles were respectively transferred to an ice bath to stop fermentation and then opened to measure medium pH using a pH meter (Metrhom pH meter, Model 691). Then, each bottle content was filtered (42 μm pore size) and a 5 ml sample of each filtrate bottle was taken and acidified with 5 ml of 0.2 N HCl and frozen at -20°C. for analyzing VFA by gas chromatography, 4 mL of each filtrate bottle were stabilized with 4 mL meta-phosphoric acid Liquid effluent was collected in flasks containing a solution of H2SO4 to maintain pH values below 2, and samples were taken for volatile fatty acids The filtrated residual was oven dried (60 °C for 48 h) and used to calculate in vitro dry matter and organic matter disappearances.
Results and Discussion: DM and OM digestibility for all treatments increased during incubation. Control treatment had lowest DM digestibility. Changes in OM digestibility was in same manner with DM digestibility in all treatments and control had lowest OM digestibility significantly. In comparison with initial pH, treatments control, Propionibacterium fredrinrichii, enterococcus faecium and mixed of Propionibacterium fredrinrichii + Enterococcus faecium had decreased 0.91, 0.73, 0.76 and 0.58 unit, respectively. NH3-N concentration after 2h after incubation was low in all treatments and increased during incubation, but for control it was low in compare with other treatments during incubation. Adding bacterial culture had significantly effect on VFA concentration. There was significantly difference among treatments on acetate: propionate ratio and treatments 2 and 4 had lowest ratio.
Conclusion: Generally, DM and OM digestibility and VFA concentration can be affected positively and significantly by live bacterial cultures additives. These changes would be associated with the stabilization of rumen pH and provide favorable conditions for microorganisms’activity in the rumen.


1. Akay, V., and R. G. Dado. 2001. Effects of propionibacterium strain P5 on in-vitro volatile fatty acids production and digestibility of fiber and starch. Turkish Journal of Veterinary and Animal Sciences, 25: 635-642.
2. Aviles, I. 1999. The use of DH42, a Propionibacterium for the prevention of lactic acidosis in cattle. M.S. Diss., Michigan State Univ., East Lansing.
3. Bach, A., S. Calsamiglia, and M. D. Stern. 2004. Nitrogen metabolism in the rumen. Journal of Dairy Science, 88 (E Suppl.):E9-E21.
4. Chesson, A., and J. Wallace. 1996. Biotechnology in animal feeds and animal feeding. Part 3: Microbial feed additives for ruminants. Feed Compounder, 16: 14-17.
5. Fuller, R. 1989. Probiotics in man and animals. Journal of Applied Bacteriology, 66: 365-78.
6. Ghorbani, G. R., D. P. Morgavi, K. A. Beauchemin, and J. A. Z. Leedle. 2002. Effects of bacterial direct-fed microbials on ruminal fermentation, blood variables and the microbial populations of feedlot cattle. Journal of Animal Science, 80:1977-1986.
7. Goad, D. W., C. L. Goad, and T. G. Nagaraja. 1998. Ruminal microbial and fermentative changes associated with experimentally induced sub-acute acidosis in steers. Journal of Animal Science, 76:234-241.
8. Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some applications). Agric. Handbook 379. ARS, USDA, Washington, DC.
9. Jones, R. J., and R. G. Megaritty. 1986. Successful transfer of DHP-degrading bacteria from Hawaiian goats to Australian ruminants to overcome the toxicity of Leucaena. Australian Veterinary Journal, 63:259-262.
10. Jouany, J. P. 1994. Methods of manipulating the microbial Metabolism in the rumen. Annales De Zootechnie, 43: 49-62.
11. Kmet, V., H. J. Flint, and R. J. Wallace. 1993. Probiotics and manipulation of rumen development and function. Archives of Animal Nutrition, 44:1-10.
12. Krehbiel, C. R., S. R. Rust, G. Zhang, and S. E. Gilliland. 2003. Bacterial direct-fed microbialsin ruminant diets: Performance response and mode of action. Journal of Animal Science, 81: E120-E132.
13. Kung, Jr. L., and A. O. Hession. 1995. Preventing in vitro lactate accumulation in ruminal fermentation by inoculation with Megasphaera elsdenii. Journal of Animal Science, 73:250-256.
14. Leng, R. A., and J. V. Nolan. 1984. Nitrogen metabolism in the rumen. Journal of Dairy Science, 67:1072-1089.
15. Mackie, R. I., and F. M. C. Gilchrist. 1979. Changes in lactate-producing and lactate-utilizing bacteria in relation to pH in the rumen of sheep during stepwise adaptation to a high-concentrate diet. Applied and Environmental Microbiology, 67:422-430.
16. Martin, S. A., and D. J. Nisbet.1991. Symposium: Direct fed microbials and rumen fermentation. Journal of Dairy Science, 75:1736-1744.
17. Mayne, C. S. 1990. An evaluation of an inoculant of lactobacillus plantarum as an addative for grass silage foe dairy cattle. Animal Production, 39: 65-76.
18. McAllister, T. A., and K. J. Cheng. 1996. Microbial strategies in the ruminal digestion of cereal grains. Animal Feed Science and Technology, 62: 29-36.
19. McAllister, T. A., Selinger, L. B., McMahon, L. R., Bae, H. D., Lysyke, T. J., Oosting, S. J., Chang, K. J. 1995. Intake, digestibility, and aerobic stability of barley silage inoculated with mixtures of Lactobacillus plantarum and Enterococcus faecium. Canadian Journal of Animal Science, 75: 425-432.
20. Nagaraja, T. G., C. J. Newbold, C. J. Van Nevel, and D. I. D. Emeyer, 1997. Manipulation of ruminal fermentation. Pages 523–632 in the rumen microbial ecosystem. P. N. Hobson and C. S. Stewart, ed. Blackie Acad. and Prof., London.
21. Newbold, C. J. 1995. Microbial feed additives for ruminants. In: R.J. Wallace and A. Chesson (Ed.) Biotechnology in Animal Feeds and Animal Feeding. Pp 259- 278.
22. Nisbet, D. J., and S. A. Martin. 1994. Factors affecting L-lactate utilization by Selenomonas ruminantium. Journal of Animal Science, 72: 1355-1361.
23. Nocek, J. E., W. P. Kautz, J. A. Z. Leedle, and J. G. Allman. 2002. Ruminal supplementation of direct-fed microbials on diurnal pH variation and in situ digestion in dairy cattle. Journal of Dairy Science, 85:429-433.
24. Oshio, S., I. Tahata, and H. Minato. 1987. Effect of diets differing in ratios of roughage to concentrate on microflora in the rumen heifers. The Journal of General and Applied Microbiology, 33: 99-111.
25. Oude Elferink, S. J. W. H., J. Krooneman, C. Gottschal, J. S. F. Spoelstra, F. Faber, and F. Driehuis. 2001. Anaerobic conversion of lactic acid to acetic acid and 1, 2 propanediol by Lactobacillus buchneri. Applied and Environmental Microbiology, 67(1): 125-132.
26. Overton, T. R., J. K. Drackley, C. J. Ottemann Abbamonte, A. D. Beaulieu, L. S. Emmert, and J. H Clark. 1999. Substrate utilization for hepatic gluconeogenesis is altered by increased glucose demand in ruminants. Journal of Animal Science, 77:1940-1951.
27. Russel, J. B., J. D. O Connor, D. G. Fox, P. J. Van Soet and C. J. Sniffer. 1992. A net carbohydrate and protein system for evaluating cattle diets. I. Ruminal fermentation. Journal of Animal Science, 70: 3551-3561.
28. Russell, J. B. 1998. The importance of pH in the regulation of ruminal acetate to propionate ratio and methane production in vitro. Journal of Dairy Science, 81: 3222-3230.
29. Slyter, L. L. 1976. Influence of acidosis on Rumen Function. Journal of Animal Science, 43:910-929.
30. Swinney-Floyd, D., B. A. Gardner, F. N. Owens, T. Rehberger, and T. Parrott. 1999. Effect of inoculation with either strain P-63 alone or in combination with Lactobacillus acidophilus LA53545 on performance of feedlot cattle. Journal of Animal Science, 77(Suppl. 1):77.
31. Van Soest, P. J., J. B.Robertson, and B. A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74:3583-3597.
32. Van Soest, P. J. 1994. Nutritional ecology of the ruminant, 2nd ed. Cornell university press, Ithaca, NY.
33. Williams, P. E. V., and C. J. Newbold. 1990. Rumen probiotics: The effects of nove microorganisms on rumen fermentation and ruminant productivity. In: Haresing W. and Cole D. J. A. (Ed) Recent advances in animal nutrition. pp 211-227. Buttersworth, London.
34. Wolin, M. J. 1988. A theoretical rumen fermentation balance. Journal of Dairy Science, 43:1452-1459.
35. Yang, W. Z., K. A. Beauchemin, D. D. Vedres, G. R. Ghorbani, D. Colombatto, and D. P. Morgavi. 2004. Effects of direct-fed microbial supplementation on ruminal acidosis, digestibility, and bacterial protein synthesis in continuous culture. Animal Feed Science and Technology, 114: 179-193.
36. Yokoyama, M. T., and K. A. Johnsos. 1988. Microbiology of the Rumen and Intestine. In: D. C. Church (Ed). The Ruminant Animal: Digestive Physiology and Nutrition. Pp 125-143. Prentice-Hall, Englewood Cliffs, New Jersey.
37. Yoon, I. K., and M. D. Stern. 1995. Influence of direct-fed microbials on ruminal microbial fermentation and performance of ruminants: A review. Asian-Australas. Journal of Animal Science, 8:533-555.