Effect of Camellia Sinensis, Punica Granatum, and Quercus Persica Extracts on in vitro Fermentation Parameters

Document Type : Research Paper


1 Department of Animal Science, Faculty of Agriculture, Gonbad Kavous University, Gonbad, Iran

2 Department of Agriculture, Payame Noor University, Gonbad, Iran


This experiment was conducted to study the effects of aqueous and alcoholic extracts of Camellia sinensis (L.) Kuntze leaves (green tea), Punica granatum (pomegranate-peel), and Quercus persica Jaub. & Spach (oak) at different concentrations on ruminal fermentation, dry matter and organic matter digestibility, methane production and protozoa population using gas production method. Experimental treatments were: control, 50, 100, and 200 μg/ ml aqueous and methanolic extract of Camellia sinensis, Punica granatum, and Q. persica (19 treatments in total). Cumulative gas production was recorded at 2, 4, 6, 8, 12, 24, 36, 48, 72, and 96 h after incubation. Dry matter digestibility (DMD), organic matter digestibility (OMD), metabolizable energy (ME), pH, and short-chain fatty acids (SCFA) were calculated after 24 h incubation. Gas production at different times, methane production, and protozoa population were also measured. DMD, OMD, and pH were decreased by adding extracts. Microbial mass production (MCP) and microbial mass production efficiency (EMCP) significantly increased at a low level (50 μg/ ml) and significantly decreased at high levels of extracts containing tannins (100 and 200 μg/ ml) (P< 0.01). The treatments also increased short-chain fatty acids (SCFA), reduced methane concentration, and reduced PF and protozoa populations only at the highest levels of extracts (P<0.05).


  1. Calsamiglia S, Busquet M, Cardozo P.W, Castillejos L, Ferret A. Invited Review: Essential Oils as Modifiers of Rumen Microbial Fermentation. J Dairy Sci. 2007;90:2580-2595.
  2. Patra A.K, Saxena J. Dietary phytochemicals as rumen modifiers: a review of the effects on microbial populations. Antonie Van Leeuwenhoek. 2009;363-375.
  3. Oeztuerk H, Sagmanligil V. Role of live yeasts in rumen ecosystem. Dtsch Tierarztl Wochenschr. 2009;116:244-248.
  4. Valdez F.R, Bush L.J, Goetsch A.L, Owens F.N. Effect of steroidal sapogenins on ruminal fermentation and on production of lactating dairy cows. J Dairy Sci. 1986;69:1568-1575.
  5. Wallace R.J, Arthaud L, Newbold C.J. Influence of Yucca shidigera extract on ruminal ammonia concentrations and ruminal microorganisms. Appl Environ Microbiol. 1994;60:1762-1767.
  6. Broudiscou L, Papon Y, Broudiscou A. Effects of dry plant extract on feed degradation and proportion of rumen microbial biomass in a dual flow ferments. Anim Feed Sci Technol. 2002;101:183-189.
  7. Beauchemin K.A, McGinn S.M, Martinez T.F, McAllister T.A. Use of condensed tannin extract from quebracho trees to reduce methane emissions from cattle. J Anim Sci. 2007;85:1990-1996.
  8. Jones G.A, McAllister T.A, Muir A.D, Cheng K.J. Effects of Sainfoin (Onobrychis viciifolia Scop.) Condensed Tannins on Growth and Proteolysis by Four Strains of Ruminal Bacteria. Appl Environ Microbiol. 1994;60:1374-1378.
  9. Smith A.H, Zoetendal E, Mackie R.I. Bacterial mechanisms to overcome inhibitory effects of dietary tannins. Microb Ecol. 2005;50:197-205.
  10. McIntosh F.M, Williams P, Losa R, Wallace R.J, Beever D.A. et al. Effects of essential oils on ruminal microorganisms and their protein metabolism. Appl Environ Microbiol. 2003;69:5011-5014.
  11. Wallace R.J, McEwan N.R, McIntosh F.M, Teferedegne B, Newbold C.J. Natural Products as Manipulators of Rumen Fermentation. Asian-Australas J Anim Sci. 2002;15:1458-1468.
  12. Bodas R, Prieto N, García-González R, Andrés S, Giráldez F. et al. Manipulation of rumen fermentation and methane production with plant secondary metabolites. Anim Feed Sci Technol. 2012;176:78-93.
  13. Makkar H.P.S, Blummel M, Becker K. In Vitro Effects of and Interactions between Tannins and Saponins and Fate of Tannins in the Rumen. J Sci Food Agric. 1995;69:481-493.
  14. Makkar H.P.S. Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies to overcome detrimental effects of feeding tannin-rich feeds. Small Rumin Res. 2003;49:241-256.
  15. Williams G.M, Wang C.X, Iatropoulos M.J. Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. II. Chronic feeding studies. Food Chem Toxicol. 1990;28:799-806.
  16. Seo J.-K, Yang J.-Y, Kim H.-J, Upadhaya S, Cho W.M. et al. Effects of Synchronization of Carbohydrate and Protein Supply on Ruminal Fermentation, Nitrogen Metabolism and Microbial Protein Synthesis in Holstein Steers. Asian-Australas J Anim Sci. 2010;23.
  17. Frutos P, Hervás G, Giráldez F.J, Mantecón A.R. Review. Tannins and ruminant nutrition. Span J Agric Res. 2004;2:191.
  18. Barry T.N, Manley T.R. The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep: 2. Quantitative digestion of carbohydrates and proteins. Br J Nutr. 2007;51:493-504.
  19. Boadi D.K, Neufeld R.J. Encapsulation of tannase for the hydrolysis of tea tannins. Enzyme Microb Technol. 2001;28:590-595.
  20. Li Y, Guo C, Yang J, Wei J, Xu J. et al. Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chem. 2006;96:254-260.
  21. Jami E, Shabtay A, Nikbachat M, Yosef E, Miron J. et al. Effects of adding a concentrated pomegranate-residue extract to the ration of lactating cows on in vivo digestibility and profile of rumen bacterial population. J Dairy Sci. 2012;95:5996-6005.
  22. Yousef Elahi M, Rouzbehan Y. Characteriztion of Quercus persica, Quercus infectoria and Quercus libani as ruminant feeds. Anim Feed Sci Technol. 2008;140:78-89.
  23. Shimada T, Saitoh T, Sasaki E, Nishitani Y, Osawa R. Role of tannin-binding salivary proteins and tannase-producing bacteria in the acclimation of the Japanese wood mouse to acorn tannins. J Chem Ecol. 2006;32:1165-1180.
  24. Chan E.W.C, Lim Y.Y, Chew Y.L. Antioxidant activity of Camellia sinensis leaves and tea from a lowland plantation in Malaysia. Food Chem. 2007;102:1214-1222.
  25. Kowalski R. Silphium L. extracts – composition and protective effect on fatty acids content in sunflower oil subjected to heating and storage. Food Chem. 2009;112:820-830.
  26. Gessner M, Steiner D. Acid Butanol Assay for Proanthocyanidins (Condensed Tannins). 2005;107-114.
  27. Menke K.H. Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Livest Res Rural Dev. 1988;28:7-55.
  28. Menke K, Raab L, Salewski A, Steingass H, Fritz D. et al. The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor in vitro. J Agric Sci. 1979;93:217-222.
  29. Theodorou M.K, Williams B.A, Dhanoa M.S, McAllan A.B, France J. A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Anim Feed Sci Technol. 1994;48:185-197.
  30. Ørskov E, McDonald I. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J Agric Sci. 1979;92:499-503.
  31. Makkar H.P. Recent advances in the in vitro gas method for evaluation of nutritional quality of feed resources. Int J Livest Prod. 2004;160:55-88.
  32. Blümmel M, Makkar H, Becker K. In vitro gas production: a technique revisited. J Anim Physiol Anim Nutr. 1997;77:24-34.
  33. Fievez V, Babayemi O, Demeyer D. Estimation of direct and indirect gas production in syringes: A tool to estimate short chain fatty acid production that requires minimal laboratory facilities. Anim Feed Sci Technol. 2005;123:197-210.
  34. Jayanegara A, Togtokhbayar N, Makkar H.P, Becker K. Tannins determined by various methods as predictors of methane production reduction potential of plants by an in vitro rumen fermentation system. Anim Feed Sci Technol. 2009;150:230-237.
  35. Dehority B.A. Rumen microbiology. Nottingham University Press Nottingham. 2003.
  36. SAS. SAS. in S.U.s. Guide., ed., SAS Institute Inc., Cary, NC. 2003.
  37. Goetsch A.L, Owens F.N. Effects of sarsaponin on digestion and passage rates in cattle fed medium to low concentrate. J Dairy Sci. 1985;68:2377-2384.
  38. Lu C.D., Jorgensen N.A. Alfalfa saponins affect site and extent of nutrient digestion in ruminants. J Nutr. 1987;117:919-927.
  39. Yanez Ruiz D, Moumen A, Martin Garcia A, Molina Alcaide E. Ruminal fermentation and degradation patterns, protozoa population, and urinary purine derivatives excretion in goats and wethers fed diets based on two-stage olive cake: Effect of PEG supply. J Anim Sci. 2004;82:2023-2032.
  40. Min B, Attwood G, Reilly K, Sun W, Peters J. et al. Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep. Can J Microbiol. 2002;48:911-921.
  41. Osakwe I.I, Steingass H, Drochner W. Effect of dried Elaeis guineense supplementation on nitrogen and energy partitioning of WAD sheep fed a basal hay diet. Anim Feed Sci Technol. 2004;117:75-83.
  42. Bhatta R, Uyeno Y, Tajima K, Takenaka A, Yabumoto Y. et al. Difference in the nature of tannins on in vitro ruminal methane and volatile fatty acid production and on methanogenic archaea and protozoal populations. J Dairy Sci. 2009;92:5512–5522.
  43. Yildiz S, Kaya I, Unal Y, Elmali D.A, Kaya S. et al. Digestion and body weight change in Tuj lambs receiving oak (Quercus hartwissiana) leaves with and without PEG. Anim Feed Sci Technol. 2005;122:159-172.
  44. Sharifi A, Chaji M. Effects of processed recycled poultry bedding with tannins extracted from pomegranate peel on the nutrient digestibility and growth performance of lambs. S Afr J Anim Sci. 2019;49:290-300.
  45. Abarghuei M, Rouzbehan Y, Salem A, Zamiri M. Nutrient digestion, ruminal fermentation and performance of dairy cows fed pomegranate peel extract. Livest Sci. 2013;157:452-461.
  46. Jolazadeh A, Dehghan-Banadaky M, Rezayazdi K. Effects of soybean meal treated with tannins extracted from pistachio hulls on performance, ruminal fermentation, blood metabolites and nutrient digestion of Holstein bulls. Anim Feed Sci Technol. 2015;203:33-40.
  47. Russell J.B, Wilson D.B. Why are ruminal cellulolytic bacteria unable to digest cellulose at low pH? J Dairy Sci. 1996;79:1503-1509.
  48. Silanikove N, Landau S., Or D, Kababya D, Bruckental I. et al. Analytical approach and effects of condensed tannins in carob pods (Ceratonia siliqua) on feed intake, digestive and metabolic responses of kids. Livest Sci. 2006;99:29-38.
  49. Motamedi H, Azizi A, Ahmadi M. Nutritive value of treated Quercus infectoria and Quercus libani leaves with the tannin‐degrading bacterium Klebsiella pneumoniae for ruminant feeding in vitro. J Appl Microbiol. 2019;127:1339-1348.
  50. Nunez-Hernandez G, Wallace J, Holechek J, Galyean M, Cardenas M. Condensed tannins and nutrient utilization by lambs and goats fed low-quality diets. J Anim Sci. 1991;69:1167-1177.
  51. Bhatta R, Uyeno Y, Tajima K, Takenaka A, Yabumoto Y. et al. Difference in the nature of tannins on in vitro ruminal methane and volatile fatty acid production and on methanogenic archaea and protozoal populations. J Dairy Sci. 2009;92:5512-5522.
  52. Hundal J, Wadhwa M, Bakshi M. Effect of herbal feed additives containing saponins on rumen fermentation pattern. Indian J Anim. Sci. 2020;90:237-243.
  53. Goel G, Makkar H.P.S, Becker K. Effects of Sesbania sesban and Carduus pycnocephalus leaves and Fenugreek (Trigonella foenum-graecum L.) seeds and their extracts on partitioning of nutrients from roughage- and concentrate-based feeds to methane. Anim Feed Sci Technol. 2008;147:72-89.
  54. Benaouda M, González-Ronquillo M, Appuhamy J, Kebreab E, Molina L. et al. Development of mathematical models to predict enteric methane emission by cattle in Latin America. Livest Sci. 2020;241:104177.
  55. Castro-Montoya J, Makkar H, Becker K. Chemical composition of rumen microbial fraction and fermentation parameters as affected by tannins and saponins using an in vitro rumen fermentation system. Can J Anim Sci. 2011;91:433-448.
  56. Makkar H.P, Sen S, Blümmel M, Becker K. Effects of fractions containing saponins from Yucca schidigera, Quillaja saponaria, and Acacia auriculoformis on rumen fermentation. J Agric Food Chem. 1998;46:4324-4328.
  57. Stewart E.K, Beauchemin K.A, Dai X, MacAdam J.W, Christensen R.G. et al. Effect of tannin-containing hays on enteric methane emissions and nitrogen partitioning in beef cattle1. J Anim Sci. 2019;97:3286-3299.
  58. Hu W.-L, Liu J.-X, Ye J.-A, Wu Y.-M, Guo Y.-Q. Effect of tea saponin on rumen fermentation in vitro. Anim Feed Sci Technol. 2005;120:333-339.
  59. Makkar H.P.S, Blümmel M, Becker K. Formation of complexes between polyvinyl pyrrolidones or polyethylene glycols and tannins, and their implication in gas production and true digestibility in in vitro techniques. Br J Nutr. 1995;73:897-913.
  60. Wina E, Muetzel S, Hoffmann E, Makkar H, Becker K. Saponins containing methanol extract of Sapindus rarak affect microbial fermentation, microbial activity and microbial community structure in vitro. Anim Feed Sci Technol. 2005;121:159-174.
  61. Kondo M, Kita K, Yokota H.o. Effects of tea leaf waste of green tea, oolong tea, and black tea addition on sudangrass silage quality and in vitro gas production. J Sci Food Agric. 2004;84:721-727.
  62. Aghamohamadi N, Hozhabri F, Alipour D. Effect of oak acorn (Quercus persica) on ruminal fermentation of sheep. Small Rumin Res. 2014;120:42-50.
  63. Van Soest P. Nutritional ecology of the ruminant. Cornell Univ. Press, Ithaca, NY. 1994.
  64. Denninger T, Schwarm A, Birkinshaw A, Terranova M, Dohme-Meier F. et al. Immediate effect of Acacia mearnsii tannins on methane emissions and milk fatty acid profiles of dairy cows. Anim Feed Sci Technol. 2020;114388.
  65. Beauchemin K.A, Janzen H.H, Little S.M, McAllister T.A, McGinn S.M. Life cycle assessment of greenhouse gas emissions from beef production in western Canada: A case study. Agric. Syst. 2010;103:371-379.
  66. Johnson K.A., Johnson D.E. Methane emissions from cattle. J Anim Sci. 1995;73:2483-2492.
  67. Morgavi D, Forano E, Martin C, Newbold C. Microbial ecosystem and methanogenesis in ruminants. Animal. 2010;4:1024-1036.
  68. Benchaar C, McAllister T, Chouinard P. Digestion, ruminal fermentation, ciliate protozoal populations, and milk production from dairy cows fed cinnamaldehyde, quebracho condensed tannin, or Yucca schidigera saponin extracts. J Dairy Sci. 2008;91:4765-4777.
  69. Raghuvansi S, Tripathi M, Mishra A, Chaturvedi O, Prasad R. et al. Feed digestion, rumen fermentation and blood biochemical constituents in Malpura rams fed a complete feed-block diet with the inclusion of tree leaves. Small Rumin Res. 2007;71:21-30.
  70. Hristov A.N, Grandeen K.L, Ropp J.K, Greer D. Effect of Yucca schidigera-based surfactant on ammonia utilization in vitro, and in situ degradability of corn grain. Anim Feed Sci Technol. 2004;115:341-355.