pp 1–10 | Cite as

Vaginal microbiome analysis of buffalo (Bubalus bubalis) during estrous cycle using high-throughput amplicon sequence of 16S rRNA gene

  • Mahalingam Srinivasan 
  • Dharumadurai Dhanasekaran 
  • Govindaraju ArchunanEmail author


Animal hosts and the microbiota that symbiotically inhabit them benefit each other reciprocally. The microbiota potentially benefit from their animal hosts by increasing the microbial communities through communication signals. Recent investigations reveal novel and important connections between microbiota of an animal and the latter’s social behavior. It has been proposed that i) the bacteria produce odorous metabolites for their hosts for purpose of communication and ii) variation in chemical signals of the host is a product of variation in the inhabiting bacterial communities. Though the vaginal microbiome contributes greatly to aspects of reproduction in cattle, including producing odor, there is only a very limited knowledge about the composition of the microbiome during various phases of the estrous cycle. Hence, the present study was undertaken to evaluate and compare microbial diversity in the vagina of buffalo during various phases of the estrous cycle by adopting high throughput sequencing methods. Microbes belonging to Proteobacteria (98.19%), Actinobacteria (1.28%), Firmicutes (0.34%), and Bacteroidetes (0.18%) were present during the pre-estrus phase. Those belonging to Proteobacteria (40.22%), Firmicutes (26.17%), Actinobacteria (18.66%), Bacteroidetes (13.29%), and Tenericutes (0.03%) were present during the estrus phase. During the di-estrus phase, Proteobacteria (85.22%), Actinobacteria (5.89%), Firmicutes (3.82%), Bacteroidetes (0.21%), and Tenericutes (4.7%) were present. Our data provide evidence for diversity of vaginal microbiota, which may be greatly influenced by the host’s physiology. The increased abundance of unassigned taxa in the vagina may play a significant biological role in the estrous cycle of the host. The study also revealed that the bacteria inhabiting buffalo vagina are distinct during different phases of the estrous cycle. It is suggested that the physiological and biochemical processes that occur in the vagina during the various phases of estrous cycle could play significant roles in determining the microbial composition, and provide vital information about microbial community structure in the buffalo vagina during the estrous cycle. Knowledge about the vaginal microbiota would contribute to improved management of buffalo reproduction.


Metagenomics Microbiome Buffalo Vagina Estrus Next generation sequencing (NGS) 



We thank Dr. A. Arun (Veterinary Gynecologist), Subject Matter Specialist, Animal Science KVK, Karur, Tamilnadu, India, for his support during sample collection. MS thanks the organizers of 9th International Symbiosis Society Congress (ISS, U.S.A) for providing international travel Scholarship for participation on ISS Congress 2018. We thank Prof. M.A. Akbarsha, National College (Autonomous), Tiruchirappalli, for critical reading of the manuscript. GA acknowledges the UGC, New Delhi, for the award of UGC-BSR Faculty Fellowship. Instrumentation facility availed from UGC-SAP, DST-FIST & PURSE, Government of India, is gratefully acknowledged.

Supplementary material

13199_2018_595_MOESM1_ESM.doc (438 kb)
ESM 1 (DOC 437 kb)


  1. Aagaard K, Riehle K, Ma J, Segata N, Mistretta TA, Coarfa C, Raza S, Rosenbaum S, Van den Veyver I, Milosavljevic A, Gevers D, Huttenhower C, Petrosino J, Versalovic J (2012) A metagenomic approach to characterization of the vaginal microbiome signature in pregnancy. PLoS One 7(6):e36466. CrossRefGoogle Scholar
  2. Archunan G, Rajanarayanan S, Karthikeyan K (2014) Cattle pheromones. In: Mucignat-Caretta C (ed) Neurobiology of chemical communication, 1st eds. CRC Press, New York, pp 461–479CrossRefGoogle Scholar
  3. Ata A, Turutoglu H, Kale M, Gulay MS, Pehlivanoglu F (2010) Microbial flora of normal and abnormal cervical mucous discharge associated with reproductive performance of cows and heifers in estrus. Asian Australas J Anim Sci 23(8):1007–1012CrossRefGoogle Scholar
  4. Boris S, Barbes C (2000) Role played by lactobacilli in controlling the population of vaginal pathogens. Microbes Infect 2(5):543–546. CrossRefGoogle Scholar
  5. Chaban B, Links MG, Jayaprakash TP, Wagner EC, Bourque DK, Lohn Z, Albert AYK, van Schalkwyk J, Reid G, Hemmingsen SM, Hill JE, Money DM (2014) Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle. Microbiome 2(1):23–34. CrossRefGoogle Scholar
  6. Clemmons BA, Reese ST, Dantas FG, Franco GA, Smith TP, Adeyosoye OI, Pohler KG, Myer PR (2017) Vaginal and uterine bacterial communities in postpartum lactating cows. Front Microbiol 8:1047. CrossRefGoogle Scholar
  7. El-Jakee JK, Ahmed WM, El-Seedy FR, Abd El-Moez SI (2008) Bacterial profile of the genital tract in female-buffalo during the different reproductive stages. Glob Vet 2(1):7–14Google Scholar
  8. FAO (2014) FAOSTAT Database. Food and Agriculture Organization of the United Nations, Rome, Italy. Accessed 14 Jan 2014
  9. Fettweis JM, Serrano MG, Sheth NU, Mayer CM, Glascock AL, Brooks JP, Jefferson KK, Buck GA (2012) Species-level classification of the vaginal microbiome. BMC Genomics 13(8):S17–S19. Google Scholar
  10. Fredricks DN (2011) Molecular methods to describe the spectrum and dynamics of the vaginal microbiota. Anaerobe 17(4):191–195. CrossRefGoogle Scholar
  11. Gladysheva IV, Cherkasov SV, Khlopko YA (2017) Antibacterial activities of metabolites from Corynebacterium spp. strains isolated from reproductive tract of a healthy woman against human pathogenic bacteria. Int J Pharm Bio Sci 8:549–556. CrossRefGoogle Scholar
  12. Handelsman J (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68(4):669–685. CrossRefGoogle Scholar
  13. Hassan M, Essam T, Megahed S (2018) Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples. Braz J Microbiol 49:S1–S8. CrossRefGoogle Scholar
  14. Izard MK (1983) Pheromones and reproduction in domestic animals. In: Pheromones and reproduction in mammals, 1st (Ed) Elsevier, 2012 253–285)Google Scholar
  15. Jami E, Mizrahi I (2012) Composition and similarity of bovine rumen microbiota across individual animals. PloS one 7(3):e33306. CrossRefGoogle Scholar
  16. Karthikeyan K, Archunan G (2013) Gas chromatographic mass spectrometric analysis of estrus specific volatile compounds in buffalo vaginal mucus after initial sexual foreplay. J Buffalo Sci 2(1):1–7Google Scholar
  17. Keverne EB (1976) Sex attractants in primates. J Soc Cosmet Chem 27(6):257–269Google Scholar
  18. Kim M, Kim J, Kuehn LA, Bono JL, Berry ED, Kalchayanand N, Freetly HC, Benson AK, Wells JE (2014) Investigation of bacterial diversity in the feces of cattle fed different diets. J Anim Sci 92(2):683–694. CrossRefGoogle Scholar
  19. Laguardia-Nascimento M, Branco KMGR, Gasparini MR, Giannattasio-Ferraz S, Leite LR, Araujo FMG, de Matos Salim AC, Nicoli JR, de Oliveira GC, Barbosa-Stancioli EF (2015) Vaginal microbiome characterization of Nellore cattle using metagenomic analysis. PLoS One 10(11):e0143294. CrossRefGoogle Scholar
  20. Lamont RF, Sobel JD, Akins RA, Hassan SS, Chaiworapongsa T, Kusanovic JP, Romero R (2011) The vaginal microbiome: new information about genital tract flora using molecular based techniques. BJOG 118(5):533–549. CrossRefGoogle Scholar
  21. Larsen B, Markovetz AJ, Galask RP (1977) Relationship of vaginal cytology to alterations of the vaginal microflora of rats during the estrous cycle. Appl Environ Microbiol 33(3):556–562Google Scholar
  22. Li Y, Hu X, Yang S, Zhou J, Zhang T, Qi L, Sun X, Fan M, Xu S, Cha M, Zhang M, Lin S, Liu S, Hu D (2017) Comparative analysis of the gut microbiota composition between captive and wild Forest musk deer. Front Microbiol 8:1705. CrossRefGoogle Scholar
  23. Lin C, Raskin L, Stahl DA (1997) Microbial community structure in gastrointestinal tracts of domestic animals: comparative analyses using rRNA-targeted oligonucleotide probes. FEMS Microbiol Ecol 22(4):281–294. CrossRefGoogle Scholar
  24. Lorenzen E, Kudirkiene E, Gutman N, Grossi AB, Agerholm JS, Erneholm K, Skytte C, Dalgaard MD, Bojesen AM (2015) The vaginal microbiome is stable in prepubertal and sexually mature Ellegaard Göttingen Minipigs throughout an estrous cycle. Vet Res 46(1):125. CrossRefGoogle Scholar
  25. MacIntyre DA, Chandiramani M, Lee YS, Kindinger L, Smith A, Angelopoulos N, Lehne B, Arulkumaran S, Brown R, Teoh TG, Holmes E (2015) The vaginal microbiome during pregnancy and the postpartum period in a European population. Sci Rep 5:8988. CrossRefGoogle Scholar
  26. Malmuthuge N, Griebel PJ (2014) Taxonomic identification of commensal bacteria associated with the mucosa and digesta throughout the gastrointestinal tracts of preweaned calves. Appl Environ Microbiol 80(6):2021–2028. CrossRefGoogle Scholar
  27. Mao S, Zhang M, Liu J, Zhu W (2015) Characterising the bacterial microbiota across the gastrointestinal tracts of dairy cattle: membership and potential function. Sci Rep 5:16116. CrossRefGoogle Scholar
  28. Ming L, Yi L, Hasi S, He J, Hai L, Wang Z, Guo F, Qiao X (2017) Comparative analysis of fecal microbial communities in cattle and Bactrian camels. PLoS One 12(3):e0173062. CrossRefGoogle Scholar
  29. Nakano FY, Leao RDBF, Esteves SC (2015) Insights into the role of cervical mucus and vaginal pH in unexplained infertility. Med Express 2(2):1–8CrossRefGoogle Scholar
  30. Nesengani LT, Wang J, Yang Y, Yang L, Lu W (2017) Unravelling vaginal microbial genetic diversity and abundance between Holstein and Fleckvieh cattle. RSC Adv 7(88):56137–56143. CrossRefGoogle Scholar
  31. Ponnusamy L, Xu N, Nojima S, Wesson DM, Schal C, Apperson CS (2008) Identification of bacteria and bacteria-associated chemical cues that mediate oviposition site preferences by Aedes aegypti. Proc Natl Acad Sci U S A 105(27):9262–9267. CrossRefGoogle Scholar
  32. Qiao H, Zhang L, Shi H, Song Y, Bian C (2018) Astragalus affects fecal microbial composition of young hens as determined by 16S rRNA sequencing. AMB Express 8(1):70. CrossRefGoogle Scholar
  33. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, Karlebach S, Gorle R, Russell J, Tacket CO, Brotman RM (2011) Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci U S A 108:4680–4687. CrossRefGoogle Scholar
  34. Sankar R, Archunan G (2004) Flehmen response in bull: role of vaginal mucus and other body fluids of bovine with special reference to estrus. Behav Process 67:81–86. CrossRefGoogle Scholar
  35. Sankar R, Archunan G (2011) Gas chromatographic/mass spectrometric analysis of volatile metabolites in bovine vaginal fluid and assessment of their bioactivity. Int J Anal Chem 011:256106–256107. Google Scholar
  36. Santos TM, Bicalho RC (2012) Diversity and succession of bacterial communities in the uterine fluid of postpartum metritic, endometritic and healthy dairy cows. PLoS ONE 7:e53048. CrossRefGoogle Scholar
  37. Silva MALd, Medeiros Z, Soares CRP, da Silva ED, Miranda-Filho DB, de Melo FL (2014) A comparison of four DNA extraction protocols for the analysis of urine from patients with visceral leishmaniasis. Rev Soc Bras Med Trop 47(2):193–197Google Scholar
  38. Singh I, Balhara AK (2016) New approaches in buffalo artificial insemination programs with special reference to India. Theriogenology 86(1):194–199. CrossRefGoogle Scholar
  39. Stevenson J, Hill S, Bridges G, Larson J, Lamb G (2015) Progesterone status, parity, body condition, and days postpartum before estrus or ovulation synchronization in suckled beef cattle influence artificial insemination pregnancy outcomes. J Anim Sci 93:2111–2123. CrossRefGoogle Scholar
  40. Swartz JD, Lachman M, Westveer K, O’Neill T, Geary T, Kott RW, Berardinelli JG, Hatfield PG, Thomson JM, Roberts A, Yeoman CJ (2014) Characterization of the vaginal microbiota of ewes and cows reveals a unique microbiota with low levels of lactobacilli and near-neutral pH. Front Vet Sci 1:19. CrossRefGoogle Scholar
  41. Teeling H, Glockner FO (2012) Current opportunities and challenges in microbial metagenome analysis-a bioinformatic perspective. Brief Bioinform 13(6):728–742. CrossRefGoogle Scholar
  42. Theis KR, Schmidt TM, Holekamp KE (2012) Evidence for a bacterial mechanism for group-specific social odors among hyenas. Sci Rep 2:615. CrossRefGoogle Scholar
  43. Theis KR, Venkataraman A, Dycus JA, Koonter KD, Schmitt-Matzen EN, Wagner AP, Holekamp KE, Schmidt TM (2013) Symbiotic bacteria appear to mediate hyena social odors. Proc Natl Acad Sci U S A 110(49):19832–19837. CrossRefGoogle Scholar
  44. Wang WL, Xu SY, Ren ZG, Tao L, Jiang JW, Zheng SS (2015) Application of metagenomics in the human gut microbiome. World J Gastroenterol 21(3):803–814. CrossRefGoogle Scholar
  45. Wang J, Sun C, Liu C, Yang Y, Lu W (2016) Comparison of vaginal microbial community structure in healthy and endometritis dairy cows by PCR-DGGE and real-time PCR. Anaerobe 38:1–6. CrossRefGoogle Scholar
  46. Wang J, Li C, Nesengani LT, Gong Y, Zhang S, Lu W (2017) Characterization of vaginal microbiota of endometritis and healthy sows using high-throughput pyrosequencing of 16S rRNA gene. Microb Pathog 111:325–330. CrossRefGoogle Scholar
  47. Yassin AF, Hupfer H, Siering C, Schumann P (2011) Comparative chemotaxonomic and phylogenetic studies on the genus Arcanobacterium Collins et al. 1982 emend. Lehnen et al. 2006: proposal for Trueperella gen. nov. and emended description of the genus Arcanobacterium. International Journal of Systematic and Evolutionary Microbiology 61(6):1265–1274Google Scholar
  48. Zeng Y, Zeng D, Ni X, Zhu H, Jian P, Zhou Y, Xu S, Lin Y, Li Y, Yin Z, Pan K (2017) Microbial community compositions in the gastrointestinal tract of Chinese Mongolian sheep using Illumina MiSeq sequencing revealed high microbial diversity. AMB Express 7(1):75. CrossRefGoogle Scholar
  49. Zhao W, Wang Y, Liu S, Huang J, Zhai Z, He C, Ding J, Wang J, Wang H, Fan W, Zhao J (2015) The dynamic distribution of porcine microbiota across different ages and gastrointestinal tract segments. PLoS One 10(2):p.e0117441.
  50. Ziemer CJ (2013) Newly cultured bacteria with broad diversity isolated from 8 weeks continuous culture enrichments of cow feces on complex polysaccharides. Appl Environ Microbiol 80:574–585. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Mahalingam Srinivasan 
    • 1
  • Dharumadurai Dhanasekaran 
    • 2
  • Govindaraju Archunan
    • 1
    Email author
  1. 1.Department of Animal ScienceBharathidasan UniversityTiruchirappalliIndia
  2. 2.Department of MicrobiologyBharathidasan UniversityTiruchirappalliIndia

Personalised recommendations