In this study, we compared the effects of two diets containing different isoflavone concentrations on the isoflavone transfer from feed into milk and on the rumen microbiota in lactating dairy cows. The on-farm experiment was conducted on twelve lactating Czech Fleckvieh x Holstein cows divided into two groups, each with similar mean milk yield. Twice daily, cows were individually fed a diet based on maize silage, meadow hay and supplemental mixture. Control group (CTRL) received the basal diet while the experimental group (EXP) received the basal diet supplemented with 40% soybean isoflavone extract. The average daily isoflavone intake in the EXP group (16 g/day) was twice as high as that in the CTRL group (8.4 g/day, P<0.001). Total isoflavone concentrations in milk from the CTRL and EXP groups were 96.89 and 276.07 μg/L, respectively (P<0.001). Equol concentrations in milk increased from 77.78 μg/L in the CTRL group to 186.30 μg/L in the EXP group (P<0.001). The V3-4 region of bacterial 16S rRNA genes was used for metagenomic analysis of the rumen microbiome. The experimental cows exhibited fewer OTUs at a distance level of 0.03 compared to control cows (P<0.05) and reduced microbial richness compared to control cows based on the calculated Inverse Simpson and Shannon indices. Non-metric multidimensional scaling analysis showed that the major contributor to separation between the experimental and control groups were changes in the representation of bacteria belonging to the phyla Bacteroidetes, Proteobacteria, Firmicutes, and Planctomycetes. Surprisingly, a statistically significant positive correlation was found only between isoflavones and the phyla Burkholderiales (r = 0.65, P<0.05) and unclassified Betaproteobacteria (r = 0.58, P<0.05). Previous mouse and human studies of isoflavone effects on the composition of gastrointestinal microbial populations generally report similar findings.

Department of Biochemistry Faculty of Science Masaryk University Brno Czech Republic

Department of Information Technologies Faculty of Informatics Masaryk University Brno Czech Republic

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Welkie DG, Stevenson DM, Weimer PJ. ARISA analysis of ruminal bacterial community dynamics in lactating dairy cows during the feeding cycle. Anaerobe. 2010; 16(2): 94–100. 10.1016/j.anaerobe.2009.07.002 PubMed DOI

Kamra DN, Pathak NN. Improvement in livestock productivity by use of probiotics: A review. Indian J Anim Sci. 2005; 75(1): 128–134.

Kong Y, Teather R, Forster R. Composition, spatial distribution, and diversity of the bacterial communities in the rumen of cows fed different forages. FEMS Microbiol Ecol. 2010; 74(3): 612–622. 10.1111/j.1574-6941.2010.00977.x PubMed DOI

Ishler V, Heinrichs A, Vafga G. From feed to milk: Understanding rumen function.: Pennsylvania State University Extension Circular 422, University Park, PA; 1996.

Hobson P, Stewart C. The Rumen Microbial Ecosystem. 2nd ed Chapman and Hall, London; 1997.

Tajima K, Aminov RI, Nagamine T, Matsui H, Nakamura M, Benno Y. Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl Environ Microbiol. 2001; 67(6): 2766–2774. PubMed PMC

Krizova L, Richter M, Trinacty J, Riha J, Kumprechtova D. The effect of feeding live yeast cultures on ruminal pH and redox potential in dry cows as continuously measured by a new wireless device. Czech J Anim Sci. 2011; 56(1): 37–45.

Marden JP, Bayourthe C, Enjalbert F, Moncoulon R. A new device for measuring kinetics of ruminal pH and redox potential in dairy cattle. J Dairy Sci. 2005; 88(1): 277–281. PubMed

Stark A, Madar Z. Phytoestrogens: A review of recent findings. J Pediatr Endocrinol Metab. 2002; 15(5): 561–572. PubMed

Duffy C, Perez K, Partridge A. Implications of phytoestrogen intake for breast cancer. CA Cancer J Clin. 2007; 57(5): 260–277. PubMed

Bhathena SJ, Velasquez MT. Beneficial role of dietary phytoestrogens in obesity and diabetes. Am J Clin Nutr. 2002; 76(6): 1191–1201. PubMed

Coward L, Barnes NC, Setchell KDR, Barnes S. Genistein, daidzein, and their beta-glycoside conjugates—antitumor isoflavones in soybean foods from american and asian diets. J Agricult Food Chem. 1993; 41(11): 1961–1967.

Setchell KDR, Brown NM, Lydeking-Olsen E. The clinical importance of the metabolite equol—A clue to the effectiveness of soy and its isoflavones. J Nutr. 2002; 132(12): 3577–3584. PubMed

Kostelac D, Rechkemmer G, Briviba K. Phytoestrogens modulate binding response of estrogen receptors alpha and beta to the estrogen response element. J AgricFood Chem. 2003; 51: 7632–7635. PubMed

Morito K, Hirose T, Kinjo J, Hirakawa T, Okawa M, Nohara T, et al. Interaction of phytoestrogens with estrogen receptors α and β. Biol Pharm Bull. 2001; 24: 351–356. PubMed

Turner R, Baron T, Wolffram S, Minihane AM, Cassidy A, Rimbach G, et al. Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein. Free Radical Res. 2004; 38: 209–216. PubMed

Lund TD, Munson DJ, Haldy ME, Setchell KDR, Lephart ED, Handa RJ. Equol is a novel anti-androgen that inhibits prostate growth and hormone feedback. Biol Reprod. 2004; 70: 1188–1195. PubMed

Mustonen EA, Tuori M, Saastamoinen I, Taponen J, Wahala K, Saloniemi H, et al. Equol in milk of dairy cows is derived from forage legumes such as red clover. British J Nutr. 2009; 102(11): 1552–1556. PubMed

Steinshamn H, Purup S, Thuen E, Hansen-Moller J. Effects of clover-grass silages and concentrate supplementation on the content of phytoestrogens in dairy cow milk. J Dairy Sci. 2008; 91(7): 2715–2725. 10.3168/jds.2007-0857 PubMed DOI

Kalac P. Fresh and ensiled forages as a source of estrogenic equol in bovine milk: a review. Czech J Anim Sci. 2013; 58(7): 296–303.

Nakamura Y, Tsuji S, Tonogai Y. Determination of the levels of isoflavonoids in soybeans and soy-derived foods and estimation of isoflavonoids in the Japanese daily intake. J AOAC Int. 2000; 83(3): 635–650. PubMed

Klejdus B, Vitamvasova-Sterbova D, Kuban V. Identification of isoflavone conjugates in red clover (Trifolium pratense) by liquid chromatography-mass spectrometry after two-dimensional solid-phase extraction. Anal Chim Acta. 2001; 450(1–2): 81–97.

Lundh T. Metabolism of estrogenic isoflavones in domestic-animals. Proc Soc Exp Biol Med. 1995; 208(1): 33–39. PubMed

Setchell KDR, Borriello SP, Hulme P, Kirk DN, Axelson M. Nonsteroidal estrogens of dietary origin—possible roles in hormone-dependent disease. Am J Clin Nutr. 1984; 40(3): 569–578. PubMed

Wang XL, Shin KH, Hur HG, Kim SI. Enhanced biosynthesis of dihydrodaidzein and dihydrogenistein by a newly isolated bovine rumen anaerobic bacterium. J Biotech. 2005; 115(3): 261–269. PubMed

Zhao H, Wang X-L, Zhang H-L, Li C-D, Wang S-Y. Production of dihydrodaidzein and dihydrogenistein by a novel oxygen-tolerant bovine rumen bacterium in the presence of atmospheric oxygen. Appl Microbiol Biotechnol. 2011; 92(4): 803–813. 10.1007/s00253-011-3278-3 PubMed DOI

Edwards JE, McEwan NR, Travis AJ, Wallace RJ. 16S rDNA library-based analysis of ruminal bacterial diversity. Antonie Van Leeuwenhoek. 2004; 86(3): 263–281. PubMed

Deng W, Xi D, Mao H, Wanapat M. The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review. Mol Biol Rep. 2008; 35(2): 265–274. PubMed

Stahl DA, Flesher B, Mansfield HR, Montgomery L. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol. 1988; 54(5): 1079–1084. PubMed PMC

Hur HG, Beger RD, Heinze TM, Lay JO, Freeman JP, Dore J, et al. Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein. Arch Microbiol. 2002; 178(1): 8–12. PubMed

Schoefer L, Mohan R, Braune A, Birringer M, Blaut M. Anaerobic C-ring cleavage of genistein and daidzein by Eubacterium ramulus. FEMS Microbiol Lett. 2002; 208(2): 197–202. PubMed

Blaut M, Schoefer L, Braune A. Transformation of flavonoids by intestinal microorganisms. Int J Vitam Nutr Res. 2003; 73(2): 79–87. PubMed

Tsangalis D, Ashton JF, McGill AEJ, Shah NP. Enzymic transformation of isoflavone phytoestrogens in soymilk by beta-glucosidase-producing bifidobacteria. J Food Sci. 2002; 67(8): 3104–3113.

Hur HG, Lay JO, Beger RD, Freeman JP, Rafii F. Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin. Arch Microbiol. 2000; 174(6): 422–428. PubMed

Kim M, Morrison M, Yu Z. Phylogenetic diversity of bacterial communities in bovine rumen as affected by diets and microenvironments. Folia Microbiol. 2011; 56(5): 453–458. PubMed

Association of Analytical Communities. Official Methods of Analysis, Association of Official Analytical Chemists, 14th ed Arlington, Virginia, USA, 1984, p. 1141; 1984.

Vansoest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci. 1991; 74(10): 3583–3597. PubMed

Goering H, Van Soest P. Forage Fiber Analyses In: Agriculture Handbook No 379, Agricultural Research Service, USDA, Washington, DC, USA; 1970. pp. 20.

Conway E. Microdiffusion Analysis and Volumetric Error.: 5th ed Crosby Lockwood, London, U.K; 1984.

Trinacty J, Krizova L, Schulzova V, Hajslova J, Hanus O. The effect of feeding soybean-derived phytoestogens on their concentration in plasma and milk of lactating dairy cows. Arch Anim Nutr. 2009; 63(3): 219–229.

Kasparovska J, Krizova L, Lochman J, Dadakova K, Kasparovsky T. Soybean-derived isoflavone determination in rumen fluid and milk by HPLC-MS-(TOF). J Chromatogr Sci. 2016; In press. 10.1093/chromsci/bmw033 PubMed DOI

Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 2013; 41(1). 10.1093/nar/gks808 PubMed DOI PMC

Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD. Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Appl Environ Microbiol. 2013; 79(17): 5112–5120. 10.1128/AEM.01043-13 PubMed DOI PMC

Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol. 2005; 71(12): 8228–8235. PubMed PMC

McMurdie PJ, Holmes S. phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data. PLoS One. 2013; 8(4). e61217 10.1371/journal.pone.0061217 PubMed DOI PMC

Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013; 41(D1): D590–D6. 10.1093/nar/gks1219 PubMed DOI PMC

Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011; 27(16): 2194–2200. 10.1093/bioinformatics/btr381 PubMed DOI PMC

Flachowsky G, Huenerberg M, Meyer U, Kammerer DR, Carle R, Goerke M, et al. Isoflavone concentration of soybean meal from various origins and transfer of isoflavones into milk of dairy cows. J Verbrauch Lebensm. 2011; 6(4): 449–456.

Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr. 1997; 17: 353–381. PubMed

Jami E, Israel A, Kotser A, Mizrahi I. Exploring the bovine rumen bacterial community from birth to adulthood. ISME J. 2013; 7(6): 1069–1079. 10.1038/ismej.2013.2 PubMed DOI PMC

Singh KM, Jisha TK, Reddy B, Parmar N, Patel A, Patel AK, et al. Microbial profiles of liquid and solid fraction associated biomaterial in buffalo rumen fed green and dry roughage diets by tagged 16S rRNA gene pyrosequencing. Mol Biol Rep. 2015; 42(1): 95–103. 10.1007/s11033-014-3746-9 PubMed DOI

Vetrovsky T, Baldrian P. The Variability of the 16S rRNA Gene in Bacterial Genomes and Its Consequences for Bacterial Community Analyses. PLoS One. 2013; 8(2): e57923 10.1371/journal.pone.0057923 PubMed DOI PMC

Křížová L, Třináctý J, Hajšlová J, Havlíková Š. The effect of technological processing on the content of isoflavones in bovine milk and dairy products In: Soybean—Applications and Technology, 1st ed In Tech, Croatia; 2011. pp.95–110.

Seymour WM, Campbell DR, Johnson ZB. Relationships between rumen volatile fatty acid concentrations and milk production in dairy cows: a literature study. Anim Feed Sci Technol. 2005; 119: 155–169.

Antignac JP, Cariou R, Le Bizec B, Andre F. New data regarding phytoestrogens content in bovine milk. Food Chem. 2004; 87(2): 275–281.

Andersen C, Nielsen TS, Purup S, Kristensen T, Eriksen J, Soegaard K, et al. Phyto-oestrogens in herbage and milk from cows grazing white clover, red clover, lucerne or chicory-rich pastures. Animal. 2009; 3(8): 1189–1195. 10.1017/S1751731109004613 PubMed DOI

Hojer A, Adler S, Purup S, Hansen-Moller J, Martinsson K, Steinshamn H, et al. Effects of feeding dairy cows different legume-grass silages on milk phytoestrogen concentration. J Dairy Sci. 2012; 95(8): 4526–4540. 10.3168/jds.2011-5226 PubMed DOI

Hoikkala A, Mustonen E, Saastamolnen I, Jokela T, Taponen J, Hannu S, et al. High levels of equol in organic skimmed Finnish cow milk. Mol Nutr Food Res. 2007; 51(7): 782–786. PubMed

Adler SA, Purup S, Hansen-Moller J, Thuen E, Gustavsson AM, Steinshamn H. Phyto-oestrogens and their metabolites in milk produced on two pastures with different botanical compositions. Livest Sci. 2014; 163: 62–68.

Nozière P, Glasser F, Sauvant D. In vivo production and molar percentages of volatile fatty acids in the rumen: a quantitative review by an empirical approach. Animal. 2011; 5(3): 403–414. 10.1017/S1751731110002016 PubMed DOI

Woclawek-Potocka I, Mannelli C, Boruszewska D, Kowalczyk-Zieba I, Wasniewski T, Skarzynski DJ. Diverse Effects of Phytoestrogens on the Reproductive Performance: Cow as a Model. Int J Endocrinol. 2013. 10.1155/2013/650984 PubMed DOI PMC

Zhengkang H, Wang G, Yao W, Zhu W. Isoflavonic Phytoestrogens—New Prebiotics for Farm Animals: a Review on Research in China. Curr Issues Intest Microbiol. 2006; 7: 53–60. PubMed

Yang G, Chen W, Chen J, Han Z. Detection of testosterone levels in rumen of male buffalo and research of its dynamic variations. J Nanjing Agric Univ. 1998(20):82–86.

Chen J, Yang G, Han Z. Effect of daidzein on serum testosterone and rumen digestion, metabolism in ruminants. Jiangsu Agric Res. 1999(20):17–19.

Wang XL, Hur HG, Lee JH, Kim KT, Kim SI. Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated anaerobic human intestinal bacterium. Appl Environ Microbiol. 2005; 71(1): 214–219. PubMed PMC

Hur HG, Beger RD, Heinze TM, Lay JO, Freeman JP, Dore J, et al. Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein. Arch Microbiol. 2002; 178(1): 8–12. PubMed

Decroos K, Vanhemmens S, Cattoir S, Boon N, Verstraete W. Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions. Arch Microbiol. 2005; 183(1): 45–55. PubMed

Yao W, Zhu WY, Han ZK, Akkermans ADL, Williams B, Tamminga S. Analysis of rumen bacterial diversity of goat by denaturing gradient gel electrophoresis and 16S rDNA sequencing. Sci Agric Sinica. 2004; 37: 1374–1378.

Clavel T, Fallani M, Lepage P, Levenez F, Mathey J, Rochet V, et al. Isoflavones and functional foods alter the dominant intestinal microbiota in postmenopausal women. J Nutr. 2005; 135(12): 2786–2792. PubMed

Vanhoutte T, Huys G, De Brandt E, Swings J. Temporal stability analysis of the microbiota in human feces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers. FEMS Microbiol Ecol. 2004; 48(3): 437–446. 10.1016/j.femsec.2004.03.001 PubMed DOI

De Boever P, Deplancke B, Verstraete W. Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. J Nutr. 2000; 130(10): 2599–2606. PubMed

Menon R, Watson SE, Thomas LN, Allred CD, Dabney A, Azcarate-Peril MA, et al. Diet Complexity and Estrogen Receptor beta Status Affect the Composition of the Murine Intestinal Microbiota. Appl Environ Microbiol. 2013; 79(18): 5763–5773. 10.1128/AEM.01182-13 PubMed DOI PMC

Isoflavones