Analyze Diet
Animals : an open access journal from MDPI2026; 16(4); 618; doi: 10.3390/ani16040618

Characterization of the Endometrial Microbiota of Healthy Mares Across the Estrous Cycle.

Abstract: In the past, bacteria detected in the mare's uterus were generally interpreted as a sign of endometritis, since the uterus was considered a sterile environment. This assumption has been challenged by the introduction of culture-independent molecular techniques, particularly 16S rRNA amplicon sequencing, which have demonstrated that healthy mares harbor an endometrial microbiota. The aim of this study was to characterize the endometrial microbiota of healthy mares and to determine whether microbial composition differs between estrus and diestrus. Endometrial samples were collected from eleven healthy Standardbred mares during estrus and diestrus and analyzed by sequencing the V1-V2 region of the bacterial 16S rRNA gene. A total of 24 bacterial phyla and 599 genera were identified. At the phylum level, Firmicutes, Proteobacteria, Bacteroidota, and Actinobacteriota accounted for most of the relative abundance, while the most abundant genera were , , , , , , and . Alpha diversity was significantly higher during estrus, likely due to hormonally mediated changes in cervical opening and local immunity. Beta diversity analyses showed substantial overlap between estrus and diestrus samples. The phase of the cycle had a weak effect on microbiota structure, while inter-individual differences between mares explained a larger proportion of the observed variation. These findings suggest that the uterine microbiota of healthy mares is largely stable across the estrous cycle, with phase-dependent and mare-specific fluctuations in microbial composition.
Publication Date: 2026-02-15 PubMed ID: 41751079PubMed Central: PMC12937484DOI: 10.3390/ani16040618Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
  • Journal Article

Cite This Article

APA
Donato GG, Necchi D, Gionechetti F, Ala U, Nebbia P, Robino P, Stella MC, Vandaele H, Pallavicini A, Nervo T. (2026). Characterization of the Endometrial Microbiota of Healthy Mares Across the Estrous Cycle. Animals (Basel), 16(4), 618. https://doi.org/10.3390/ani16040618

Publication

ISSN: 2076-2615
NlmUniqueID: 101635614
Country: Switzerland
Language: English
Volume: 16
Issue: 4
PII: 618

Researcher Affiliations

Donato, Gian Guido
  • Department of Veterinary Sciences, University of Turin, 10095 Grugliasco, Italy.
Necchi, Denis
  • Keros Embryo Transfer Center, 8980 Passendale, Belgium.
Gionechetti, Fabrizia
  • Department of Life Sciences, University of Trieste, 34127 Trieste, Italy.
Ala, Ugo
  • Department of Veterinary Sciences, University of Turin, 10095 Grugliasco, Italy.
Nebbia, Patrizia
  • Department of Veterinary Sciences, University of Turin, 10095 Grugliasco, Italy.
Robino, Patrizia
  • Department of Veterinary Sciences, University of Turin, 10095 Grugliasco, Italy.
Stella, Maria Cristina
  • Department of Veterinary Sciences, University of Turin, 10095 Grugliasco, Italy.
Vandaele, Hilde
  • Keros Embryo Transfer Center, 8980 Passendale, Belgium.
Pallavicini, Alberto
  • Department of Life Sciences, University of Trieste, 34127 Trieste, Italy.
Nervo, Tiziana
  • Department of Veterinary Sciences, University of Turin, 10095 Grugliasco, Italy.

Conflict of Interest Statement

G.G.D., F.G., U.A., P.N., P.R., M.C.S., A.P., T.N. declare no conflicts of interest; D.N. and H.V. are employees of Keros.

References

This article includes 42 references
  1. Samper J.C. Breeding the Problem Mare by Artificial Insemination. AAEP Proc. 2008;54:408–413.
  2. Holyoak G.R., Premathilake H.U., Lyman C.C., Sones J.L., Gunn A., Wieneke X., DeSilva U. The Healthy Equine Uterus Harbors a Distinct Core Microbiome plus a Rich and Diverse Microbiome That Varies with Geographical Location. Sci. Rep. 2022;12:14790. doi: 10.1038/s41598-022-18971-6.
    doi: 10.1038/s41598-022-18971-6pmc: PMC9427864pubmed: 36042332google scholar: lookup
  3. Gil-Miranda A., Macnicol J., Orellana-Guerrero D., Samper J.C., Gomez D.E. Reproductive Tract Microbiota of Mares. Vet. Sci. 2024;11:324. doi: 10.3390/vetsci11070324.
    doi: 10.3390/vetsci11070324pmc: PMC11281493pubmed: 39058008google scholar: lookup
  4. Aurich C. Reproductive Cycles of Horses. Anim. Reprod. Sci. 2011;124:220–228. doi: 10.1016/j.anireprosci.2011.02.005.
  5. Pozor M.A. Ultrasound Evaluation of the Reproductive Tract in Mares: Why Ultrasonography Is an Essential Component of Daily Practice. AAEP Proc. 2017;63:340–356.
  6. Marth C.D., Firestone S.M., Glenton L.Y., Browning G.F., Young N.D., Krekeler N. Oestrous Cycle-Dependent Equine Uterine Immune Response to Induced Infectious Endometritis. Vet. Res. 2016;47:110. doi: 10.1186/s13567-016-0398-x.
    doi: 10.1186/s13567-016-0398-xpmc: PMC5101692pubmed: 27825391google scholar: lookup
  7. Evans M.J., Hamer J.M., Gason L.M., Graham C.S., Ausbury A.C., Irvine C.H.G. Clearance of Bacteria and Non-Antigenic Markers Following Intra-Uterine Inoculation into Maiden Mares: Effect of Steroid Hormone Environment. Theriogenology. 1986;26:37–50. doi: 10.1016/0093-691X(86)90110-X.
    doi: 10.1016/0093-691X(86)90110-Xpubmed: 16726168google scholar: lookup
  8. Gil-Miranda A., Caddey B., Orellana-Guerrero D., Smith H., Samper J.C., Gomez D.E. Vaginal and Uterine Microbiota of Healthy Maiden Mares during Estrus. Vet. Sci. 2024;11:323. doi: 10.3390/vetsci11070323.
    doi: 10.3390/vetsci11070323pmc: PMC11281598pubmed: 39058007google scholar: lookup
  9. Canisso I.F., Segabinazzi L.G.T.M., Fedorka C.E. Persistent Breeding-Induced Endometritis in Mares—A Multifaceted Challenge: From Clinical Aspects to Immunopathogenesis and Pathobiology. Int. J. Mol. Sci. 2020;21:1432. doi: 10.3390/ijms21041432.
    doi: 10.3390/ijms21041432pmc: PMC7073041pubmed: 32093296google scholar: lookup
  10. Trifanescu O.G., Trifanescu R.A., Mitrica R.I., Bran D.M., Serbanescu G.L., Valcauan L., Marinescu S.A., Gales L.N., Tanase B.C., Anghel R.M. The Female Reproductive Tract Microbiome and Cancerogenesis: A Review Story of Bacteria, Hormones, and Disease. Diagnostics. 2023;13:877. doi: 10.3390/diagnostics13050877.
  11. Mallott E.K., Borries C., Koenig A., Amato K.R., Lu A. Reproductive Hormones Mediate Changes in the Gut Microbiome during Pregnancy and Lactation in Phayre’s Leaf Monkeys. Sci. Rep. 2020;10:9961. doi: 10.1038/s41598-020-66865-2.
    doi: 10.1038/s41598-020-66865-2pmc: PMC7305161pubmed: 32561791google scholar: lookup
  12. Quereda J.J., Barba M., Mocé M.L., Gomis J., Jiménez-Trigos E., García-Muñoz Á., Gómez-Martín Á., González-Torres P., Carbonetto B., García-Roselló E. Vaginal Microbiota Changes During Estrous Cycle in Dairy Heifers. Front. Vet. Sci. 2020;7:371. doi: 10.3389/fvets.2020.00371.
    doi: 10.3389/fvets.2020.00371pmc: PMC7350931pubmed: 32719814google scholar: lookup
  13. Gohil P., Patel K., Purohit K., Chavda D., Puvar A., Suthar V.S., Patil D.B., Joshi M., Joshi C.G. Metagenomic Analysis of Bacterial Communities during Estrous Cycle in Bos Indicus. Trop. Anim. Health Prod. 2022;54:118. doi: 10.1007/s11250-022-03119-5.
    doi: 10.1007/s11250-022-03119-5pubmed: 35226189google scholar: lookup
  14. Vomstein K., Reider S., Böttcher B., Watschinger C., Kyvelidou C., Tilg H., Moschen A.R., Toth B. Uterine Microbiota Plasticity during the Menstrual Cycle: Differences between Healthy Controls and Patients with Recurrent Miscarriage or Implantation Failure. J. Reprod. Immunol. 2022;151:103634. doi: 10.1016/j.jri.2022.103634.
    doi: 10.1016/j.jri.2022.103634pubmed: 35550495google scholar: lookup
  15. Heil B.A., van Heule M., Thompson S.K., Kearns T.A., Beckers K.F., Oberhaus E.L., King G., Daels P., Dini P., Sones J.L. Metagenomic Characterization of the Equine Endometrial Microbiome during Anestrus. J. Equine Vet. Sci. 2024;140:105134. doi: 10.1016/j.jevs.2024.105134.
    doi: 10.1016/j.jevs.2024.105134pubmed: 38909766google scholar: lookup
  16. Barba M., Martínez-Boví R., Quereda J.J., Mocé M.L., Plaza-Dávila M., Jiménez-Trigos E., Gómez-Martín Á., González-Torres P., Carbonetto B., García-Roselló E. Vaginal Microbiota Is Stable throughout the Estrous Cycle in Arabian Mares. Animals. 2020;10:2020. doi: 10.3390/ani10112020.
    doi: 10.3390/ani10112020pmc: PMC7692283pubmed: 33153053google scholar: lookup
  17. Samper J.C. Ultrasonographic Appearance and the Pattern of Uterine Edema to Time Ovulation in Mares. AAEP Proc. 1997;43:189–191.
  18. Donato G.G., Appino S., Bertero A., Poletto M.L., Nebbia P., Robino P., Varello K., Bozzetta E., Vincenti L., Nervo T. Safety and Effects of a Commercial Ozone Foam Preparation on Endometrial Environment and Fertility of Mares. J. Equine Vet. Sci. 2023;121:104222. doi: 10.1016/j.jevs.2023.104222.
    doi: 10.1016/j.jevs.2023.104222pubmed: 36623580google scholar: lookup
  19. Donato G.G., Nebbia P., Stella M.C., Scalas D., Necchi D., Bertero A., Romano G., Bergamini L., Poletto M.L., Peano A., et al. In Vitro Effects of Different Ozone Preparations on Microorganisms Responsible for Endometritis in the Mare. Theriogenology. 2024;219:132–137. doi: 10.1016/j.theriogenology.2024.02.011.
  20. Banchi P., Bertero A., Gionechetti F., Corrò M., Spagnolo E., Donato G.G., Pallavicini A., Rota A. The Vaginal Microbiota of Healthy Female Cats. Theriogenology. 2024;224:134–142. doi: 10.1016/j.theriogenology.2024.05.021.
  21. Walker S.P., Barrett M., Hogan G., Flores Bueso Y., Claesson M.J., Tangney M. Non-Specific Amplification of Human DNA Is a Major Challenge for 16S RRNA Gene Sequence Analysis. Sci. Rep. 2020;10:16356. doi: 10.1038/s41598-020-73403-7.
    doi: 10.1038/s41598-020-73403-7pmc: PMC7529756pubmed: 33004967google scholar: lookup
  22. Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J.A., Holmes S.P. DADA2: High-Resolution Sample Inference from Illumina Amplicon Data. Nat. Methods. 2016;13:581–583. doi: 10.1038/nmeth.3869.
    doi: 10.1038/nmeth.3869pmc: PMC4927377pubmed: 27214047google scholar: lookup
  23. Beckers K.F., Liu C.C., Gomes V.C.L., Schulz C.J., Childers G.W., Fedorka C.E., Sones J.L. Effects of Intra-Uterine Ceftiofur on the Equine Uterine Microbiome. Vet. Sci. 2025;12:837. doi: 10.3390/vetsci12090837.
    doi: 10.3390/vetsci12090837pmc: PMC12474077pubmed: 41012762google scholar: lookup
  24. Virendra A., Gulavane S.U., Ahmed Z.A., Reddy R., Chaudhari R.J., Gaikwad S.M., Shelar R.R., Ingole S.D., Thorat V.D., Khanam A., et al. Metagenomic Analysis Unravels Novel Taxonomic Differences in the Uterine Microbiome between Healthy Mares and Mares with Endometritis. Vet. Med. Sci. 2024;10:e1369. doi: 10.1002/vms3.1369.
    doi: 10.1002/vms3.1369pmc: PMC10867593pubmed: 38357732google scholar: lookup
  25. Thomson P., Pareja J., Núñez A., Santibáñez R., Castro R. Characterization of Microbial Communities and Predicted Metabolic Pathways in the Uterus of Healthy Mares. Open Vet. J. 2022;12:797–805. doi: 10.5455/OVJ.2022.v12.i6.3.
    doi: 10.5455/OVJ.2022.v12.i6.3pmc: PMC9805769pubmed: 36650865google scholar: lookup
  26. Heil B.A., van Heule M., Thompson S.K., Kearns T.A., Oberhaus E.L., King G., Daels P., Dini P., Sones J.L. Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus. Vet. Sci. 2023;10:644. doi: 10.3390/vetsci10110644.
    doi: 10.3390/vetsci10110644pmc: PMC10675083pubmed: 37999467google scholar: lookup
  27. Guo L., Holyoak G.R., DeSilva U. Endometrial Microbiome in Mares with and without Clinical Endometritis. Front. Vet. Sci. 2025;12:1588432. doi: 10.3389/fvets.2025.1588432.
    doi: 10.3389/fvets.2025.1588432pmc: PMC12355929pubmed: 40822659google scholar: lookup
  28. Berg G., Rybakova D., Fischer D., Cernava T., Vergès M.C.C., Charles T., Chen X., Cocolin L., Eversole K., Corral G.H., et al. Microbiome Definition Re-Visited: Old Concepts and New Challenges. Microbiome. 2020;8:103. doi: 10.1186/s40168-020-00875-0.
    doi: 10.1186/s40168-020-00875-0pmc: PMC7329523pubmed: 32605663google scholar: lookup
  29. Galla G., Praeg N., Rzehak T., Sprecher E., Colla F., Seeber J., Illmer P., Hauffe H.C. Comparison of DNA Extraction Methods on Different Sample Matrices within the Same Terrestrial Ecosystem. Sci. Rep. 2024;14:8715. doi: 10.1038/s41598-024-59086-4.
    doi: 10.1038/s41598-024-59086-4pmc: PMC11018758pubmed: 38622248google scholar: lookup
  30. López-Aladid R., Fernández-Barat L., Alcaraz-Serrano V., Bueno-Freire L., Vázquez N., Pastor-Ibáñez R., Palomeque A., Oscanoa P., Torres A. Determining the Most Accurate 16S RRNA Hypervariable Region for Taxonomic Identification from Respiratory Samples. Sci. Rep. 2023;13:3974. doi: 10.1038/s41598-023-30764-z.
    doi: 10.1038/s41598-023-30764-zpmc: PMC9998635pubmed: 36894603google scholar: lookup
  31. Levy M., Bassis C.M., Kennedy E., Yoest K.E., Becker J.B., Bell J., Berger M.B., Bruns T.M. The Rodent Vaginal Microbiome across the Estrous Cycle and the Effect of Genital Nerve Electrical Stimulation. PLoS ONE. 2020;15:e0230170. doi: 10.1371/journal.pone.0230170.
  32. Matějková T., Dodoková A., Kreisinger J., Stopka P., Stopková R. Microbial, Proteomic, and Metabolomic Profiling of the Estrous Cycle in Wild House Mice. Microbiol. Spectr. 2024;12:e0203723. doi: 10.1128/spectrum.02037-23.
    doi: 10.1128/spectrum.02037-23pmc: PMC10846187pubmed: 38171017google scholar: lookup
  33. Lehtoranta L., Ala-Jaakkola R., Laitila A., Maukonen J. Healthy Vaginal Microbiota and Influence of Probiotics Across the Female Life Span. Front. Microbiol. 2022;13:819958. doi: 10.3389/fmicb.2022.819958.
    doi: 10.3389/fmicb.2022.819958pmc: PMC9024219pubmed: 35464937google scholar: lookup
  34. Ault T.B., Clemmons B.A., Reese S.T., Dantas F.G., Franco G.A., Smith T.P.L., Edwards J.L., Myer P.R., Pohler K.G. Bacterial Taxonomic Composition of the Postpartum Cow Uterus and Vagina Prior to Artificial Insemination. J. Anim. Sci. 2019;97:4305–4313. doi: 10.1093/jas/skz212.
    doi: 10.1093/jas/skz212pmc: PMC6776284pubmed: 31251804google scholar: lookup
  35. Kaur H., Merchant M., Haque M.M., Mande S.S. Crosstalk Between Female Gonadal Hormones and Vaginal Microbiota Across Various Phases of Women’s Gynecological Lifecycle. Front. Microbiol. 2020;11:551. doi: 10.3389/fmicb.2020.00551.
    doi: 10.3389/fmicb.2020.00551pmc: PMC7136476pubmed: 32296412google scholar: lookup
  36. Marth C.D., Young N.D., Glenton L.Y., Noden D.M., Browning G.F., Krekeler N. Deep Sequencing of the Uterine Immune Response to Bacteria during the Equine Oestrous Cycle. BMC Genom. 2015;16:934. doi: 10.1186/s12864-015-2139-3.
    doi: 10.1186/s12864-015-2139-3pmc: PMC4647707pubmed: 26572250google scholar: lookup
  37. LeBlanc M.M., Neuwirth L., Asbury A.C., Tran T., Mauragis D., Klapstein E. Scintigraphic Measurement of Uterine Clearance in Normal Mares and Mares with Recurrent Endometritis. Equine Vet. J. 1994;26:109–113. doi: 10.1111/j.2042-3306.1994.tb04346.x.
  38. Huchzermeyer S., Wehrend A., Bostedt H. Histomorphology of the Equine Cervix. Anat. Histol. Embryol. 2005;34:38–41. doi: 10.1111/j.1439-0264.2004.00565.x.
  39. Fernandes C.B., Ball B.A., Loux S.C., Boakari Y.L., Scoggin K.E., El-Sheikh Ali H., Cogliati B., Esteller-Vico A. Uterine Cervix as a Fundamental Part of the Pathogenesis of Pregnancy Loss Associated with Ascending Placentitis in Mares. Theriogenology. 2020;145:167–175. doi: 10.1016/j.theriogenology.2019.10.017.
  40. Troedsson M.H.T. Uterine Clearence and Resistance to Persistent Endometritis in the Mare. Theriogenology. 1999;52:461–471. doi: 10.1016/S0093-691X(99)00143-0.
    doi: 10.1016/S0093-691X(99)00143-0pubmed: 10734380google scholar: lookup
  41. Vanstokstraeten R., Callewaert E., Blotwijk S., Rombauts E., Crombé F., Emmerechts K., Soetens O., Vandoorslaer K., De Geyter D., Allonsius C., et al. Comparing Vaginal and Endometrial Microbiota Using Culturomics: Proof of Concept. Int. J. Mol. Sci. 2023;24:5947. doi: 10.3390/ijms24065947.
    doi: 10.3390/ijms24065947pmc: PMC10055768pubmed: 36983020google scholar: lookup
  42. Toson B., Simon C., Moreno I. The Endometrial Microbiome and Its Impact on Human Conception. Int. J. Mol. Sci. 2022;23:485. doi: 10.3390/ijms23010485.
    doi: 10.3390/ijms23010485pmc: PMC8745284pubmed: 35008911google scholar: lookup

Citations

This article has been cited 0 times.