Effects of alfalfa flavonoids on the production performance, immune system, and ruminal fermentation of dairy cows

  • Zhan, Jinshun (College of Animal Science and Technology, Yangzhou University) ;
  • Liu, Mingmei (College of Animal Science and Technology, Yangzhou University) ;
  • Su, Xiaoshuang (College of Animal Science and Technology, Yangzhou University) ;
  • Zhan, Kang (College of Animal Science and Technology, Yangzhou University) ;
  • Zhang, Chungang (College of Animal Science and Technology, Yangzhou University) ;
  • Zhao, Guoqi (College of Animal Science and Technology, Yangzhou University)
  • Received : 2016.07.28
  • Accepted : 2017.03.22
  • Published : 2017.10.01


Objective: The objective of this study was to examine the effects of alfalfa flavonoids on the production performance, immunity, and ruminal fermentation of dairy cows. Methods: The experiments employed four primiparous Holstein cows fitted with ruminal cannulas, and used a $4{\times}4$ Latin square design. Cattle were fed total mixed ration supplemented with 0 (control group, Con), 20, 60, or 100 mg of alfalfa flavonoids extract (AFE) per kg of dairy cow body weight (BW). Results: The feed intake of the group receiving 60 mg/kg BW of AFE were significantly higher (p<0.05) than that of the group receiving 100 mg/kg BW. Milk yields and the fat, protein and lactose of milk were unaffected by AFE, while the total solids content of milk reduced (p = 0.05) linearly as AFE supplementation was increased. The somatic cell count of milk in group receiving 60 mg/kg BW of AFE was significantly lower (p<0.05) than that of the control group. Apparent total-tract digestibility of neutral detergent fiber and crude protein showed a tendency to increase (0.05<$p{\leq}0.10$) with ingestion of AFE. Methane dicarboxylic aldehyde concentration decreased (p = 0.03) linearly, whereas superoxide dismutase activity showed a tendency to increase (p = 0.10) quadratically, with increasing levels of AFE supplementation. The lymphocyte count and the proportion of lymphocytes decreased (p = 0.03) linearly, whereas the proportion of neutrophil granulocytes increased (p = 0.01) linearly with increasing levels of dietary AFE supplementation. The valeric acid/total volatile fatty acid (TVFA) ratio was increased (p = 0.01) linearly with increasing of the level of AFE supplementation, the other ruminal fermentation parameters were not affected by AFE supplementation. Relative levels of the rumen microbe Ruminococcus flavefaciens tended to decrease (p = 0.09) quadratically, whereas those of Butyrivibrio fibrisolvens showed a tendency to increase (p = 0.07) quadratically in response to AFE supplementation. Conclusion: The results of this study demonstrate that AFE supplementation can alter composition of milk, and may also have an increase tendency of nutrient digestion by regulating populations of microbes in the rumen, improve antioxidant properties by increasing antioxidant enzyme activities, and affect immunity by altering the proportions of lymphocyte and neutrophil granulocytes in dairy cows. The addition of 60 mg/kg BW of AFE to the diet of dairy cows was shown to be beneficial in this study.


Flavonoids;Alfalfa;Ruminal Fermentation;Nutrient Digestibility;Dairy Cow


Supported by : National Natural Science Foundation of China


  1. Byarugaba DK. A view on antimicrobial resistance in developing countries and responsible risk factors. Int J Antimicrob Agents 2004; 24:105-10.
  2. Busquet M, Calsamiglia S, Ferret A, Kamel C. Plant extracts affect in vitro rumen microbial fermentation. J Dairy Sci 2006;89:761-71.
  3. Divyashree R, Amit KR, Francesca G, Osborn HM. Flavonoids as prospective compounds for anti-cancer therapy. Int J Biochem Cell Biol 2013;45:2821-31.
  4. Ehsan O, Norhani A, Armin O. Effects of flavonoids on rumen fermentation activity, methane production, and microbial population. BioMed Res Int 2013;2013:Article ID 349129.
  5. Kim ET, Guan LL, Lee SJ, et al. Effects of flavonoid-rich plant extracts on in vitro ruminal methanogenesis, microbial populations and fermentation characteristics. Asian-Australas J Anim Sci 2015;28:530-7.
  6. Aguiar SC, Cottica SM, Boeing JS, et al. Effect of feeding phenolic compounds from propolis extracts to dairy cows on milk production, milk fatty acid composition, and the antioxidant capacity of milk. Anim Feed Sci Technol 2014;193:148-54.
  7. Liu DY, He SJ, Jin EH, et al. Effect of daidzein on production performance and serum antioxidative function in late lactation cows under heat stress. Livest Sci 2013;152:16-20.
  8. Gao WW. Study on the bioactivity of alfalfa and the influence factors to its flavonoids and saponins [Doctor's thesis]. Beijing, China: Chinese Peking Union Medical College; 2004.
  9. Benchaar C, Petit HV, Berthiaume R, Whyte TD, Chouinard PY. Effects of addition of essential oils and monensin premix on digestion, ruminal fermentation, milk production, and milk composition in dairy cows. J Dairy Sci 2006;89:4352-69.
  10. Weatherburn MW. Phenol-hypochlorite reaction for determination of ammonia. Analyt Chem 1967;39:971-4.
  11. Guo WS, Schaefer DM, Guo XX, Ren LP, Meng QX. Use of Nitratenitrogen as a sole dietary nitrogen source to inhibit ruminal methanogenesis and to improve microbial nitrogen synthesis in vitro. Asian-Australas J Anim Sci 2009;22:542-9.
  12. Hall MB, Herejk C. Differences in yields of microbial crude protein from in vitro fermentation of carbohydrates. J Dairy Sci 2001;84:2486-93.
  13. Denman SE, Mcsweeney CS. Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol Ecol 2006;58:572-82.
  14. Stevenson DM, Weimer PJ. Dominance of Prevotella and low abundance of classical ruminal bacterial species in the bovine rumen revealed by relative quantification real-time PCR. Appl Microbiol Biotechnol 2007;75:165-74.
  15. Lan Y, Xun S, Tamminga S, et al. Real-time PCR detection of lactic acid bacteria in cecal contents of Eimeria tenella-infected broilers fed soybean oligosaccharides and soluble soybean polysaccharides. Poult Sci 2004;83:1696-702.
  16. Sylvester JT, Karnati SK, Yu Z, Morrison M, Firkins JL. Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR. J Nutr 2004;134:3378-84.
  17. Gessner DK, Koch C, Romberg FJ, et al. The effect of grape seed and grape marc meal extract on milk performance and the expression of genes of endoplasmic reticulum stress and inflammation in the liver of dairy cows in early lactation. J Dairy Sci 2015;98:8856-68.
  18. Winkler A, Gessner DK, Koch C, et al. Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows. Arch Anim Nutr 2015;69:425-41.
  19. Cant JP, Trout DR, Qiao F, et al. Milk synhtetic response of the bovine mammary gland to increase in the local concentration of arterial glucose. J Dairy Sci 2002;3:494-503.
  20. Aguiar SC, Paula EM, Yoshimura EH, et al. Effects of phenolic compounds in propolis on digestive and ruminal parameters in dairy cows. Rev Bras Zootec 2014;43:197-206.
  21. Paula EM, Samensari RB, Machado E, et al. Effects of phenolic compounds on ruminal protozoa population, ruminal fermentation, and digestion in water buffaloes. Livest Sci 2016;185:136-41.
  22. Chen DD, Chen XL, Tu Y, et al. Effects of mulberry leaf flavonoid and resveratrol on methane emission and nutrient digestion in sheep. Anim Nutr 2016;1:362-7.
  23. Seradj AR, Abecia L, Crespo J, Villalba D, Fondevila M, Balcells J. The effect of Bioflavex$^{(R)}$ and its pure flavonoid components on in vitro fermentation parameters and methane production in rumen fluid from steers given high concentrate diets. Anim Feed Sci Technol 2014;197:85-91.
  24. Park JS, Russell JB, Wilson DB. Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85. Anaerobe 2007;13:83-8.
  25. Su XS, Zhan JS, Zhan K, Liu MM, Zhao GQ. Proliferation stimulus and antioxidant effect of alfalfa flavonoids on dairy cow mammary epithelial cells cultured in vitro. Acta Pratacult Sin 2015;24:139-45.
  26. Zhu ZN, Hao ZR, Wang M, Jiang LS, Guo YQ. Effects of dietary soy isoflavone supplementation on the levels of tumor necrosis factor- ${\alpha}$ and surface-type IgA secreted by mammary mast Cells in higher lactating dairy cows during late lactation. Chinese J Anim Nutr 2011; 23:112-21.
  27. Yaghoubi SMJ, Ghorbani GR, Rahmani HR, Nikkhah A. Growth, weaning performance and blood indicators of humoral immunity in Holstein calves fed supplemental flavonoids. J Anim Physiol Anim Nutr 2008;92:456-62.
  28. Satomi Y, Daisuke U, Norihide M, et al. Dietary apigenin suppresses IgE and inflammatory cytokines production in C57BL/6N mice. J Agric Food Chem 2006;54:5203-7.
  29. Liu DY, Gu YF, Wang YX, Ding CH, Chen HL. Effects of daidzein on anti-oxidative activities and immune functions in dairy cows. Chinese J Vet Sci 2008;28:1306-9.
  30. Hedi H, Fadwa C, Kamel G, Chekir-Ghedira L. Inhibition of proinflammatory macrophage responses and lymphocyte proliferation in vitro by ethyl acetate leaf extract from Daphne gnidium. Cell Immunol 2011;267:94-101.

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