Evaluation of the urogenital microbiota of healthy cyclic bitches

Evaluation of the urogenital microbiota of healthy cyclic bitches | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the urogenital microbiota of healthy cyclic bitches Virginie Gronsfeld, Flore Brutinel, Sophie Egyptien, Charles Porsmoguer, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3955899/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Jul, 2024 Read the published version in BMC Veterinary Research → Version 1 posted 4 You are reading this latest preprint version Abstract Background : Understanding the urogenital microbiota would allow us to compare the bacterial populations in healthy and pathological conditions and assess their impact on various urogenital diseases. The aim of our research was to characterize and compare the urogenital microbiota during different phases of the estrous cycle in healthy female dogs. DNA extraction, 16S rDNA library preparation, sequencing, and informatics analysis were employed to determine the vaginal and urinary microbiota in 10 healthy beagle dogs at each phase of the cycle. Results: Alpha diversity, richness, and evenness of bacterial populations in the vagina were not significantly different across the various cycle phases. However, there was a significant difference in vaginal beta diversity between the different cycle phases, except for anestrus and diestrus. Conversely, no differences in alpha and beta diversity were observed in the urinary microbiota across the different cycle phases. Conclusions : This study demonstrate estrogenic influence on the abundance of vaginal microbiota in healthy female dogs, with no discernible influence on urinary microbiota. Additionally, it provides a comparative basis for understanding the urinary and vaginal microbiota in healthy female dogs. Vaginal microbiota urinary microbiota hormonal influence dogs Figures Figure 1 Figure 2 Figure 3 Figure 4 Background In recent years, there has been a growing interest in studying the microbiome. Prior to the advent of 16S rDNA gene sequencing, investigations into the urogenital system's microbiome, specifically the bladder and vagina – both systems influenced by sex hormones – primarily relied on culture-based techniques. Unfortunately, these techniques often failed to detect most of the resident microflora. 1 In humans, 16S rDNA gene sequencing has revealed that urine is not sterile, and samples collected via cystocentesis differ from those collected via midstream voiding. 2 16S rDNA gene sequencing, has identified the presence of various microorganisms such as Lactobacillus, Corynebacterium, Streptococcus, Actinomyces, Gardnerella, and Bifidobacterium. 3 , 4 Genome phylogenetic analysis of bacterial strains isolated from the vagina and bladder in women suggests an interconnected female urogenital microbiota that extends beyond pathogens to include health-associated commensals. 5 Sex hormones have been shown to contribute to the regulation of vaginal microbiota, which, in turn, may modify mucosal estrogen levels. 6 , 7 , 8 The vaginal microbiota varies between prepubertal, pubertal, and post-menopausal women. 9 , 10 But in most women appears to remain relatively stable throughout the menstrual cycle, with little variation in diversity and modest fluctuations in species richness. 11 Recently in veterinary medicine, the urogenital microbiome has been characterized by the 16S rDNA gene sequencing in healthy dogs. Four taxa, belonging to the phylum Pseudomonadota, Pseudomonas sp, Acinetobacter sp, Sphingobium sp and Bradyrhizobiaceae , dominated the urinary microbiota in dogs of both sexes. Considerable overlap has been observed between the vaginal and the bladder microbiota where Pseudomonas and Acinetobacter were the most abundant taxa. 12 Another recent study shown that Hydrotalea , Ralstonia , Mycoplasma , Fusobacterium and Streptoccocus were the predominant species in the vagina. 13 The vaginal microbiota of the bitches in heat was found to be the most diverse and the highest in richness among the different phases of the estrous cycle. The differences however were not statistically significant except between estrus and prepubertal stage. 13 The diversity of the vaginal microbiome in female beagles was found to continuously change with age. 14 Although the influence of sex hormones on the urinary microbiota of female dogs has not been determined yet, it has been proven that the urinary system is under the influence of the estrous cycle and the sex hormones. Indeed, in dogs, results of urodynamics studies have shown that urethral pressure, which is responsible of the urinary continence, decreased during estrus and early diestrus. 15 , 16 The purpose of our research was to characterize and compare the urogenital microbiota in the different phases of the estrous cycle in healthy bitches. Since the bladder and the vaginal microbiota are closely connected, we hypothesized that the sex hormones and the estrous cycle influences both microbiotas. Material and Methods Dogs –This prospective study was conducted on 10 intact healthy adult female Beagle dogs (ULiège Ethical agreement number: 20-2250). Dogs were included in the study if they had no signs of vaginal, systemic, or lower urinary tract diseases. Dogs that had received antibiotics, probiotics and anti-inflammatory drugs within the previous 30 days were excluded. Study design –Samples were obtained from adult dogs at the following phases of an estrous cycle: proestrus, estrus, diestrus, anestrus. Phases of the estrous cycle were identified via visual examination of the vulva, cytologic examination of a vaginal smear, and determination of plasma progesterone concentration. Urine was collected from all dogs via ante pubic cystocentesis after skin disinfection with chlorhexidine soap and alcohol to minimize dermal microbiota contamination. Urine was aliquoted for routine analysis, routine culture and 16S rRNA amplicon sequencing. After placing a sterile (UV treatment 30 minutes in a BL2 Biohazard cabinet) otoscope cone beyond the vestibule, a swab moistened with sterile saline solution was passed through the speculum onto the anterior vagina that was swabbed for 10 seconds to collect a genital sample. Negative controls consisted of saline moistened vaginal swabs passed through a sterile speculum. Urine and vaginal samples were stored at -80°C until DNA extraction. DNA extraction, 16S rDNA library preparation, sequencing and informatics analysis were performed as previously described. 17 , 18 Briefly, total bacterial DNA were extracted from the vaginal swabs and the urine with the DNEasy Blood and Tissue kit with an added bead beating step during lysis (QIAGEN Benelux BV; Antwerp, Belgium). Amplicon sequencing targeting V1V3 hypervariable regions of the 16S rDNA was performed using University of Liège GIGA sequencing facility (V3 MiSeq illumina sequencer). Sequence reads cleaning and processing were done using MOTHUR software package v1.47 and SILVA v1.38_1 16S rDNA reference database. The urinary and vaginal microbiota were analyzed separately with same protocol. Following profile analyses were performed at the genus taxonomical level. Statistical analysis –Good’s coverage index and ecological indicators, including the α-diversity (inverse Simpson’s index and Shannon Index), bacterial richness (Chao1 index) and evenness (Simpson index-based measure) were calculated with MOTHUR v1.47. Indicators differences between groups were assessed using ANOVA or non-parametric Kruskal–Wallis test when non normal distribution was observed, followed by paired post hoc tests corrected with a two-stage linear step-up procedure of Benjamini Hochberg (q threshold = 0.05) with PRISM 9.0. Beta diversity was visualized with a Bray–Curtis dissimilarity matrix-based non-parametric dimensional scaling (NMDS) model using vegan and vegan3d packages in R. Differences regarding beta diversity between cycle phases was assessed with analysis of molecular variance (AMOVA) and homogeneity of molecular variance (HOMOVA) tests using MOTHUR (using 10,000 iterations on the subsampled table) on a Bray–Curtis dissimilarity matrix. Differences were considered significant p-value < 0.05. Differential population abundance between cycle phases was evaluated using a negative binomial Wald test as implemented in the DESeq2 R package. Differences were considered significant for corrected p-value < 0.05 (Benjamini-Hochberg False Discovery Rate multitesting correction). A Mantel test was performed with the Pearson, Spearman and Kendall test to evaluate the correlation between vaginal and urinary microbiota. Results Dogs – 10 intact healthy adult female Beagle dogs were included in this study. The mean weight was 14.25 kg with (range 12-17 kg). The mean age was 6.5 years-old (range 5-9 years old). Urine analysis – The pH was between 4.5 and 8 with a urine specific gravity between 1.005-1.048. Discrete proteinuria was frequently and consistently observed in some individuals. Urinary culture – Urine cultures were mostly negative, with the exception of Streptococcus infantarius and Escherichia coli in estrus and Enterococcus Hirae in diestrus in one individual and Lactobacillus Gasse in estrus in another. Vaginal microbiota – A total of 315 genera were identified in the vaginal samples during the different cycle phases. These most abundant bacterial populations belong to the genus Fusobacterium , Porphyromas , Parvimonas and Escherichia-Shigella with a mean percentage of 33.1%, 11.5%, 7.1%, 7% and 5.8%, respectively in this global sample. To investigate the possible contaminant nature of each identified genus, a correlation test was applied between each identified genus and the bacterial abundance of the global population. The negative result (Rho -0,7972; p <0.0001) for Escherichia-Shigella suggested that it was a contaminant. Microbial population ecological indices of vaginal samples were assessed at the genus level. Results of alpha diversity, richness, and evenness of the bacterial populations throughout the different cycle phases were not significantly different (Figure 1). Group clustering testing did reveal significant overall differences between samples from the different cycle phases (Anestrus-Diestrus-Estrus-Proestrus) (p <0.0001) based on an AMOVA analysis. Paired analysis did reveal significant differences between anestrus and estrus (p <0.0001), anestrus and proestrus (p <0.0001), diestrus and estrus (p <0.0001), diestrus and proestrus (p <0.0001) and, estrus and proestrus (p <0.0002). No statistical difference was demonstrated between anestrus and diestrus. HOMOVA testing was statistically significant (p =0.0035). However, paired analysis did not yield statistically different results. Beta-diversity of vaginal microbial profile was visualized using an NMDS (non-metric dimensional scaling) model based upon a Bray-Curtis dissimilarity matrix including samples of the different cycle phases (Anestrus-Diestrus-Estrus-Proestrus), which is shown in Figure 2. The AMOVA analysis resulted in statistical differences for beta diversity between the different cycle phases and paired difference analysis was significant except for anestrus and diestrus. Then differential population abundance between cycle phases was evaluated using a negative binomial Wald test as implemented in the DESeq2. No statistically significant differential population abundance between anestrus and diestrus was observed. There was a significant differential population abundance between proestrus and estrus for 5 genera ( Prevotella , Variovorax and Porphyromonas ) comprising 2 contaminants. There was a significant differential population abundance between anestrus and estrus for 32 genera ( Parvimonas, S5.A14, Peptostreptococcae genus, Anaerovoracacaea genus, Solobacterium, Porphyromonas, Fusobacterium, Alloprevotella, Peptococcus, Fusobacteriales genus, Porphyromonadacae genus, Johnsonella , …) comprising 6 contaminants. There was a significant differential population abundance between anestrus and proestrus for 28 genera ( S5.A14a, Parvimonas, Porphyromonadacae genus, Anaerovoracaceae genus, Peptostreptococcaceae genus, Solobacterium, Alloprevotella, …) comprising 7 contaminants. There was a significant differential population abundance between diestrus and proestrus for 27 genera ( S5.A14a, Porphyromonadacae, Fusobacterium , …) comprising 10 contaminants. There was a significant differential population abundance between diestrus and estrus for 33 genera ( S5.A14a, porphyromonadacae, Fusobacterium, Bacteriodes, Peptostreptococcus, Johnsella, Parvimonas, Peptostreptococcaceae genus, Prophromonadaceae genius , …) comprising 11 contaminants (Table 1). Urinary microbiota – A total of 351 genera were identified in the urinary samples during the different cycle phases. These most abundant bacterial populations belong to the genera Escherichia-Shigella , Flavobacterium , Enterobacteriaceae , Pseudomonadaceae and Rheinheimera with a mean percentage of 67%, 6.5%, 3%, 1.3% and 1%, respectively in this global sample. Group clustering testing didn’t reveal significant differences in variation between samples from the different cycle phases (Anestrus-Diestrus-Estrus-Proestrus) based on AMOVA analysis and HOMOVA testing yielded non-statistically significant differences (Figure 3). Microbial population ecological indices of vaginal samples were assessed at the genus level. Results of alpha diversity, richness and evenness of the bacterial populations throughout the different cycle phases were not significantly different (Figure 4). No statistical correlation was observed between urinary and vaginal microbiota. Discussion The global composition of the vaginal microbiome includes bacteria, fungi, viruses, archaea, and candidate phyla radiation with bacteria communities representing the biggest percentage of the vaginal microbiome. The description of the different bacterial species can be interesting when looking for a particular species (i.e., pathogen) for example to study a pathology. In this study, we chose to describe the bacterial population according to the different genera encountered. This allowed precision while avoiding noise associated with identification by species. This best suited the purpose of our research, which was to characterize and compare the urogenital microbiota in the different phases of the estrous cycle in healthy bitches. Early studies characterizing vaginal microbiota by aerobic and anaerobic culture methods in bitches yielded E. coli and S. pseudintermedius as the most common isolates. 12 , 19 It has been suspected to be a limitation of routine culture. With the 16S rDNA gene sequencing, Hydrotalea, Ralstonia, Mycoplasma, Fusobacterium and Streptoccocus were reported to be the predominant genera in the vagina of female dogs in the study of Burton et al. 2017. 12 Mycoplasma, Pasteurellaceae family and Salmonella were identified in healthy bitches of various breed in the study of Rota et al. 2020 20 and Fusobacteria, Firmicutes, Proteobacteria, Tenericutes and Bacteroidetes were identified in beagles in the study of Hu et al. 2022. 15 The results of our study partly agree with the studies of Burton et al. 12 and Hu et al. 15 In our population, Fusobacterium, Porphyromas , Parvimonas and Escherichia-Shigella were the predominant genera. As Escherichia-Shigella was suspected to be a contaminant, like in the evaluation by bacterial culture, the remaining predominant genera in our study are Fusobacterium, Porphyromas and Parvimonas . In cyclic women, vaginal microbiota is mostly dominated by Lactobacillus crispatus, L. iners, L. gasseri or L. jensenii contrary to the observations of our study. 21 Early studies using aerobic and anaerobic culture characterized urine as sterile. 22 With the 16S rDNA gene sequencing, Phylum Pseudomonadota , Pseudomonas sp, Acinetobacter sp, Sphingobium sp and Bradyrhizobiaceae were reported to dominate the urinary microbiota in dogs in a previous study. 17 The results of our study were partly in accordance with those of the previous report. In our population, Escherichia-Shigella , Flavobacterium , Enterobacteriaceae genus , Pseudomonadaceae genus and Rheinheimera were the dominant genera. Pseudomonas sp is part of the Pseudomonadaceae family. Moreover, Lactobacillus Gasse was identified in urine at the culture and confirmed by the 16S rDNA gene sequencing in one dog but was not identified in her vaginal microbiota. Escherichia-Shigella, Enterobacteriaceae , Pseudomonadaceae and Rheinheimera are parts of the Pseudomonadota phylum. Sphingobium , Acinetobacter, Rheinheimera and Flavobacterium are bacteria that have been observed in water. Corynebacterium, Streptococcus and Actinomyces , reported as part of women urinary microbiota, are found in aqueous media, but not exclusively. 3 None of the bacteria of a woman’s urinary microbiota is therefore specific to the aqueous environment. 3 This difference between the urinary microbiota of women and bitches is currently unexplained. It can be speculated that anatomical differences may account for the discrepancy or that urinary microbiota could be different depending on the species, as reported for the intestinal microbiota. 23 To identify potential contaminant, a correlation test between the presence of the genus and the abundance of the bacterial population of the vagina was performed. Escherichia-Shigella, Comamonadaceae genus, Pseudomanas, Flavobacteriaceae genus, Enterobacterales genus, Flavobacterium, Rheinheimera, Pelomonas, Acinetobacter, saccharimonadales genus, Chryseobacterium genus, Parcubactera genus, Aeromonas, Burkholderiales genus and Paracoccus should be considered as contaminants. Interestingly, Escherichia-Shigella, Flavobacterium , Enterobacteriaceae genus and Rheinheimera , which were dominant genera in urinary microbiota, are also found in the vaginal microbiota. We supposed that this is more an overlap of both microbiota than a real contamination. In a previous study, the genital microbiome was similar to the urinary microbiota. 12 However, a correlation test let suspect that Escherichia-Shigella, Flavobacterium , Enterobacteriaceae genus and Rheinheimera were vaginal contaminants and no correlation between vaginal and urinary microbiota was statistically demonstrated in our study. Thus, a contamination of the vaginal microbiota by urine during sampling can’t be excluded. A previous study showed similar microbiota in dogs with or without recurrent urinary tract infection, which seems to discard the condition as a source of variation in the vaginal microbiota. 23 Also, contaminants may come from the animal or from the environment or food during the collection procedure. In reports, in humans, an influence of the food on the urinary and vaginal microbiota has been described. 24 , 26 Our dog population is made up of laboratory beagles fed a strictly normal or light dry food diet, as opposed to a population of healthy dogs from owners who will tend to have a more varied diet including dry food/wet food as well as table scraps. They live in a kennel with wood shavings as bedding, which is cleaned daily and changed weekly, which is very different from the classic environment of owner dogs living indoors or in outdoor parks. Therefore, the external environment in which the dogs’ lives could be more contaminated (e.g., through litter with wood chips or feces) than the conventional environment of a pet. Although urine and vaginal samples were collected in a sterile environment, contamination cannot be definitively ruled out. Urogenital microbiota analysis of a group of female dogs living in a conventional environment and comparison with those of the beagles living in a laboratory kennel would be necessary to confirm this hypothesis. Sex hormones have been demonstrated to contribute to the regulation of vaginal microbiota 6 , 7 , 8 , 11 , 25 , as it covaries with estradiol level and differs in alpha and beta diversity across the menstrual cycle of women. 25 The influence of progesterone is not well known. The results of our study showed a variation of the vaginal microbiota according to the cycle of the bitches. Alpha diversity, which corresponds to the number of species coexisting in the vagina, was not statistically different during the cycle. Absence of influence on alpha diversity in our study compared with the study of Song et al. 2020 25 may be due to the fact that we analyzed the microbiota once per cyclic phase compared with this study that did it once daily or can be due to the difference of species between dogs and women. No influence of progesterone on vaginal microbiota could be shown in our study as beta diversity did not differ between diestrus, when progesterone is highest, and anestrus, when there are no significant levels of circulating sex hormones. Indeed, estradiol-β17 concentration peaks during proestrus and decreases during estrus to be at the lowest concentration during diestrus and anestrus. In contrast, the progesterone concentration increases during the estrus to be at the highest concentration during diestrus. Progesterone concentration is low during anestrus and proestrus. 26 Therefore, the variation of beta diversity during estrogenic phases compared to diestrus and anestrus highlights the influence of estrogens on the vaginal microbiome as described in woman. 11 In women, estrogens induce a stimulation of vaginal epithelial cells proliferation, with a mid-cycle peak in intracellular glycogen levels in the vaginal mucosa and a subsequent increase in lactic acid producing microbes, such as Lactobacillus , in the vaginal milieu. 27 While similar effect of estrogens on vaginal cells proliferation exists in the dog 28 , the mechanism explaining the changes in the microbiome remain to be explored. Moreover, Lactobacillus hasn’t been identified in vaginal microbiota in our population of middle-aged females what suggests a really different vaginal environment between women and dogs contrary to the study of Hu J. et al. 2022 in which, Lactobacillus was present and increased in proportion as the dogs aged. 15 The difference between the vagina in bitches and women, with a pH of 7 and 4.5 respectively, could explain why the microbiota composition is significantly different. 20 Other factors that could influence vaginal microbiota during the cycle include the presence of blood in the environment during proestrus, which may contribute to the changes in beta-diversity by the presence of iron or increased pH. 21 The blood could also influence the vaginal microbiota by providing substrate for growth proliferation or flushing out bacteria. We postulated that estrogens could influence the urinary microbiota in bitches because urodynamics studies have shown an influence of the estrous cycle and the sex hormones on urinary system by decreasing urethral pressure during estrous and early diestrus. 15 , 29 Estrogens induce an increase in the number of alpha-adrenergic receptors and responsiveness of these receptors to sympathetic stimulation. 15 Estrogens also induce an increase in blood flow to urethral tissues 29 , which causes an increase in urethral sphincter tone. 30 Progesterone potentiates beta-adrenergic activity in urethra of female dogs, leading to relaxation and a decrease in urethral smooth muscle tone. 31 – 33 However, no influence of the cycle and thus of the estrogenic impregnation on alpha and beta diversity of the urinary microbiota could be demonstrated in this study. The absence of variation of alpha and beta diversity of the urinary microbiota may be due to the absence of change in the environment during the cycle. Indeed, if estrogens and progesterone have an indirect action on urethral pressure, there is no reported change in the urinary system structure, namely the epithelium, unlike for the vagina. The main limitations in this study were the possible presence of contaminants in the sample despite all the aseptic methods used for collection and the small number of dogs included. Another source of contamination could come from bedding in the kennel even if the litter was changed weekly and cleaned daily. However, this was a homogenous group of dogs regarding the breed, age, housing conditions and food. Conclusion The perspectives of this study are numerous. The next step will be to describe the evolution of the urinary and genital microbiota in growing healthy male and female dogs, and afterwards to study the urogenital microbiota in pathological conditions. To conclude, this study demonstrated an estrogenic influence on the abundance of vaginal microbiota in healthy female dogs and provided a comparative basis of urinary and vaginal microbiota in healthy female dogs. Declarations a. Ethics approval and consent to participate This research had the ULiège Ethical agreement number: 20-2250 b. Consent for publication Not applicable. Participants are laboratory dogs for which participation has been authorized by the ULiège Ethical agreement number: 20-2250 c. Availability of data and materials The data of sample and statistical analysis are available on request to Virginie Gronsfeld d. Competing interests The authors report there are no competing interests to declare. e. Author contributions: V.G. Wrote the Ian manuscript text F.B., S.E., C.P. Participated to the collection of data A.H. and G.D. Provided material B.T. Did the analysis of data M-L.VdW. Did the analysis of sample S.D. and S.N. reviewed the manuscript f. Funding The authors have no funding to declare. g. Acknowledgments / References Theron J, Cloete TE. Molecular techniques for determining microbial diversity and community structure in natural environments. Crit Rev Microbiol 2000; 26(1):37‐57.https://doi.org/10.1080/10408410091154174 Coffey EL, Gomez AM, Ericsson AC, Burton EN, Granick JL, Lulich JP et al. The impact of urine collection method on canine urinary microbiota detection: a cross-sectional study. BMC Microbiol 2023; 23(1):101https//doi.org/10.1186/s12866-023-02815-y Hilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, Mueller ER et al. Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. 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Am J Vet Res 2012;73:1657-1664. https//doi.org/10.2460/ajvr.73.10.1657 Raz S, Caine M, Zeigler M. The vascular component in the production of intraurethral pressure. J Urol 1972;108(1):93‐96. https//doi.org/10.1016/s0022-5347(17)60650-5 Raz S, Zeigler M, Caine M. Br. The effect of progesterone on the adrenergic receptors of the urethra. J Urol 1972;45(2):131‐5. https//doi.org/10.1111/j.1464-410x.1973.tb12129.x Caine M, Raz S. Some clinical implications of adrenergic receptors in the urinary tract. Arch Surg 1975;110(3):247‐50. https//doi.org/10.1001/archsurg.1975.01360090017003 Holt PE. Urinary incontinence in the bitch due to sphincter mechanism incompetence: prevalence in referred dogs and retrospective analysis of sixty cases. 1985, Small Anim Pract, 26:181–190. https://doi.org/10.1111/j.1748-5827.1985.tb02099.x Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Cite Share Download PDF Status: Published Journal Publication published 15 Jul, 2024 Read the published version in BMC Veterinary Research → Version 1 posted Editorial decision: Revision requested 20 Feb, 2024 Editor assigned by journal 15 Feb, 2024 Submission checks completed at journal 15 Feb, 2024 First submitted to journal 14 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3955899","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":273060988,"identity":"8aa96479-a4d6-4ae3-97bf-b161cd1b0dce","order_by":0,"name":"Virginie Gronsfeld","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3PsYrCMBzH8YRAXH7U1VC1r9Cj0Em8B3EJCHFxcxIHr9zq82QOBOrmLLjYuydwOKiTRlFwanQTLl8y/AP5kISQUOhtG5AuMYTu3YjoOaIIHGHphfBXCO9cZi9JRrasajlAZJDP/6bDLies+tk2kI+NmmSQCsKRXU+P3cN4lk2byAp5TGuL1HC1E5o5Ah77iKjl6UpmQi/9JAHyDqRxhJX0oK2fpOAqhhxDWGZjqtfgzPOXZMVK97BhP1oXxeGoF5/t1nf123iLuU/MLdyGxpKvhw2tPadDoVDof3YGE+o+Q4LUyTkAAAAASUVORK5CYII=","orcid":"","institution":"University of Liège","correspondingAuthor":true,"prefix":"","firstName":"Virginie","middleName":"","lastName":"Gronsfeld","suffix":""},{"id":273060990,"identity":"d06507c9-e105-46b7-9ada-0c700678d997","order_by":1,"name":"Flore Brutinel","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Flore","middleName":"","lastName":"Brutinel","suffix":""},{"id":273060992,"identity":"7a03f308-d3c7-4b1f-9bca-c9dbf2886863","order_by":2,"name":"Sophie Egyptien","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Sophie","middleName":"","lastName":"Egyptien","suffix":""},{"id":273060994,"identity":"515a5a75-5eaa-4472-aed7-5e9bb52b2a53","order_by":3,"name":"Charles Porsmoguer","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Charles","middleName":"","lastName":"Porsmoguer","suffix":""},{"id":273060995,"identity":"77a83c09-6b92-4dd7-a166-e86d73366280","order_by":4,"name":"Annick Hamaide","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Annick","middleName":"","lastName":"Hamaide","suffix":""},{"id":273060996,"identity":"b9489fc4-833e-40ed-9984-095aba03116e","order_by":5,"name":"Bernard Taminiau","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Bernard","middleName":"","lastName":"Taminiau","suffix":""},{"id":273060997,"identity":"6302b4b4-780b-43f9-8a8d-abef41a3e23a","order_by":6,"name":"Georges Daube","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Georges","middleName":"","lastName":"Daube","suffix":""},{"id":273060998,"identity":"668336b3-79fb-4681-a8f6-92b661d4ffbc","order_by":7,"name":"Marie-Lys Weerdt","email":"","orcid":"","institution":"Labforvet","correspondingAuthor":false,"prefix":"","firstName":"Marie-Lys","middleName":"","lastName":"Weerdt","suffix":""},{"id":273060999,"identity":"43bcc140-f2e1-4fbe-8e83-0224ab43a107","order_by":8,"name":"Stefan Deleuze","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Deleuze","suffix":""},{"id":273061000,"identity":"172d8895-d7c2-43d9-b95d-5aa657338de7","order_by":9,"name":"Stéphanie Noel","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Stéphanie","middleName":"","lastName":"Noel","suffix":""}],"badges":[],"createdAt":"2024-02-14 11:00:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3955899/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3955899/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-024-04104-w","type":"published","date":"2024-07-15T16:13:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51317459,"identity":"3fd9a0a0-0e42-4c1d-8790-d11b4128f6aa","added_by":"auto","created_at":"2024-02-19 13:12:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":304408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eScatterplots depicting alpha-diversity indices of vaginal microbiota at each phase of the cycle.\u003c/u\u003e Each dot represents a subsample. Bacterial intrinsic diversity was deduced from reciprocal Simpson Biodiversity index, bacterial genus richness from Chao1 index and bacterial genus evenness from Simpson index. No significant difference was found between groups regarding reciprocal Simpson index, population richness and Simpson derived evenness based on a Friedman test. Horizontal lines represent the mean, and error bars indicate the 95% CIs for each group and time point.\u003c/p\u003e","description":"","filename":"FigurePage1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3955899/v1/0d36101a67e69a96be4a1ef7.jpg"},{"id":51317460,"identity":"a59a52e1-ea34-419b-b08f-ff5fd6867db4","added_by":"auto","created_at":"2024-02-19 13:12:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":158534,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eNonmetric multidimensional scaling model (k=3, stress= 0.0836) based upon a Bray-Curtis dissimilarity matrix of the vaginal microbial profiles during the cycle phases\u003c/u\u003e. The dots of each color represent the different subsamples in one cycle phases. Group clustering testing did reveal significant differences in variance between samples except between anestrus and diestrus samples (p-value=0.0001) based on AMOVA analysis. HOMOVA testing yielded statistically significant differences (p =0.0035). Signification of the dots: Black anestrus, red diestrus, green estrus, blue proestrus.\u003c/p\u003e","description":"","filename":"FigurePage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3955899/v1/a354198111da6dc678a79e3d.jpg"},{"id":51317728,"identity":"0e6d8d07-ce07-47a3-a3ba-1d4ff609a290","added_by":"auto","created_at":"2024-02-19 13:20:41","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":278528,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eScatterplots depicting alpha-diversity indices of vaginal microbiota at each phase of the cycle\u003c/u\u003e. Each dot represents a subsample. Bacterial intrinsic diversity was deduced from reciprocal Simpson Biodiversity index, bacterial genus richness from Chao1 index and bacterial genus evenness from Simpson index. No significant difference was observed between the groups regarding reciprocal Simpson index, population richness and Simpson derived evenness based on a Friedman test. Horizontal lines represent the mean, and error bars indicate the 95% CIs for each group and time point.\u003c/p\u003e","description":"","filename":"FigurePage3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3955899/v1/c3dbfd18b38c7c82c695bfa2.jpg"},{"id":51317461,"identity":"df9b42f5-eb62-4e9f-927d-2a11ea002c3f","added_by":"auto","created_at":"2024-02-19 13:12:41","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003eNonmetric multidimensional scaling model (k=4, stress=0.084) based upon a Bray-Curtis dissimilarity matrix of the urinary microbial profiles during the cycle phases\u003c/u\u003e. The dots of each color represent the different subsamples in one cycle phases. Group clustering testing did not reveal significant differences in variance between samples based on AMOVA and HOMOVA. analysis. Black anestrus, red diestrus, green estrus, blue proestrus.\u003c/p\u003e","description":"","filename":"FigurePage4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-3955899/v1/8e9e833fc253906040bc4497.jpg"},{"id":61596731,"identity":"c00a1d3f-bfb9-435f-a9e9-09f90fcb429f","added_by":"auto","created_at":"2024-08-01 17:29:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1182002,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3955899/v1/0adcf760-fc3f-4005-8a33-c4c3bb79f86d.pdf"},{"id":51317463,"identity":"daeb30d2-253f-463c-a73e-9490e97d441d","added_by":"auto","created_at":"2024-02-19 13:12:41","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14070,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3955899/v1/b8a51e482199d38b38f9263d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the urogenital microbiota of healthy cyclic bitches","fulltext":[{"header":"Background","content":"\u003cp\u003eIn recent years, there has been a growing interest in studying the microbiome. Prior to the advent of 16S rDNA gene sequencing, investigations into the urogenital system's microbiome, specifically the bladder and vagina \u0026ndash; both systems influenced by sex hormones \u0026ndash; primarily relied on culture-based techniques. Unfortunately, these techniques often failed to detect most of the resident microflora.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e In humans, 16S rDNA gene sequencing has revealed that urine is not sterile, and samples collected via cystocentesis differ from those collected via midstream voiding.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e 16S rDNA gene sequencing, has identified the presence of various microorganisms such as \u003cem\u003eLactobacillus, Corynebacterium, Streptococcus, Actinomyces, Gardnerella, and Bifidobacterium.\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Genome phylogenetic analysis of bacterial strains isolated from the vagina and bladder in women suggests an interconnected female urogenital microbiota that extends beyond pathogens to include health-associated commensals.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Sex hormones have been shown to contribute to the regulation of vaginal microbiota, which, in turn, may modify mucosal estrogen levels.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e The vaginal microbiota varies between prepubertal, pubertal, and post-menopausal women.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e But in most women appears to remain relatively stable throughout the menstrual cycle, with little variation in diversity and modest fluctuations in species richness.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecently in veterinary medicine, the urogenital microbiome has been characterized by the 16S rDNA gene sequencing in healthy dogs. Four taxa, belonging to the phylum \u003cem\u003ePseudomonadota, Pseudomonas\u003c/em\u003e sp, \u003cem\u003eAcinetobacter\u003c/em\u003e sp, \u003cem\u003eSphingobium\u003c/em\u003e sp and \u003cem\u003eBradyrhizobiaceae\u003c/em\u003e, dominated the urinary microbiota in dogs of both sexes. Considerable overlap has been observed between the vaginal and the bladder microbiota where \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003eAcinetobacter\u003c/em\u003e were the most abundant taxa.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Another recent study shown that \u003cem\u003eHydrotalea\u003c/em\u003e, \u003cem\u003eRalstonia\u003c/em\u003e, \u003cem\u003eMycoplasma\u003c/em\u003e, \u003cem\u003eFusobacterium\u003c/em\u003e and \u003cem\u003eStreptoccocus\u003c/em\u003e were the predominant species in the vagina.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The vaginal microbiota of the bitches in heat was found to be the most diverse and the highest in richness among the different phases of the estrous cycle. The differences however were not statistically significant except between estrus and prepubertal stage.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e The diversity of the vaginal microbiome in female beagles was found to continuously change with age.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough the influence of sex hormones on the urinary microbiota of female dogs has not been determined yet, it has been proven that the urinary system is under the influence of the estrous cycle and the sex hormones. Indeed, in dogs, results of urodynamics studies have shown that urethral pressure, which is responsible of the urinary continence, decreased during estrus and early diestrus.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe purpose of our research was to characterize and compare the urogenital microbiota in the different phases of the estrous cycle in healthy bitches. Since the bladder and the vaginal microbiota are closely connected, we hypothesized that the sex hormones and the estrous cycle influences both microbiotas.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003eDogs \u0026ndash;This prospective study was conducted on 10 intact healthy adult female Beagle dogs (ULi\u0026egrave;ge Ethical agreement number: 20-2250). Dogs were included in the study if they had no signs of vaginal, systemic, or lower urinary tract diseases. Dogs that had received antibiotics, probiotics and anti-inflammatory drugs within the previous 30 days were excluded.\u003c/p\u003e \u003cp\u003e Study design \u0026ndash;Samples were obtained from adult dogs at the following phases of an estrous cycle: proestrus, estrus, diestrus, anestrus. Phases of the estrous cycle were identified via visual examination of the vulva, cytologic examination of a vaginal smear, and determination of plasma progesterone concentration. Urine was collected from all dogs via ante pubic cystocentesis after skin disinfection with chlorhexidine soap and alcohol to minimize dermal microbiota contamination. Urine was aliquoted for routine analysis, routine culture and 16S rRNA amplicon sequencing. After placing a sterile (UV treatment 30 minutes in a BL2 Biohazard cabinet) otoscope cone beyond the vestibule, a swab moistened with sterile saline solution was passed through the speculum onto the anterior vagina that was swabbed for 10 seconds to collect a genital sample. Negative controls consisted of saline moistened vaginal swabs passed through a sterile speculum. Urine and vaginal samples were stored at -80\u0026deg;C until DNA extraction.\u003c/p\u003e \u003cp\u003eDNA extraction, 16S rDNA library preparation, sequencing and informatics analysis were performed as previously described.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Briefly, total bacterial DNA were extracted from the vaginal swabs and the urine with the DNEasy Blood and Tissue kit with an added bead beating step during lysis (QIAGEN Benelux BV; Antwerp, Belgium). Amplicon sequencing targeting V1V3 hypervariable regions of the 16S rDNA was performed using University of Li\u0026egrave;ge GIGA sequencing facility (V3 MiSeq illumina sequencer). Sequence reads cleaning and processing were done using MOTHUR software package v1.47 and SILVA v1.38_1 16S rDNA reference database. The urinary and vaginal microbiota were analyzed separately with same protocol. Following profile analyses were performed at the genus taxonomical level.\u003c/p\u003e \u003cp\u003eStatistical analysis \u0026ndash;Good\u0026rsquo;s coverage index and ecological indicators, including the α-diversity (inverse Simpson\u0026rsquo;s index and Shannon Index), bacterial richness (Chao1 index) and evenness (Simpson index-based measure) were calculated with MOTHUR v1.47. Indicators differences between groups were assessed using ANOVA or non-parametric Kruskal\u0026ndash;Wallis test when non normal distribution was observed, followed by paired post hoc tests corrected with a two-stage linear step-up procedure of Benjamini Hochberg (q threshold\u0026thinsp;=\u0026thinsp;0.05) with PRISM 9.0.\u003c/p\u003e \u003cp\u003eBeta diversity was visualized with a Bray\u0026ndash;Curtis dissimilarity matrix-based non-parametric dimensional scaling (NMDS) model using vegan and vegan3d packages in R. Differences regarding beta diversity between cycle phases was assessed with analysis of molecular variance (AMOVA) and homogeneity of molecular variance (HOMOVA) tests using MOTHUR (using 10,000 iterations on the subsampled table) on a Bray\u0026ndash;Curtis dissimilarity matrix. Differences were considered significant p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eDifferential population abundance between cycle phases was evaluated using a negative binomial Wald test as implemented in the DESeq2 R package. Differences were considered significant for corrected p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Benjamini-Hochberg False Discovery Rate multitesting correction).\u003c/p\u003e \u003cp\u003eA Mantel test was performed with the Pearson, Spearman and Kendall test to evaluate the correlation between vaginal and urinary microbiota.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDogs \u0026ndash; 10 intact healthy adult female Beagle dogs were included in this study. The mean weight was 14.25 kg with (range 12-17 kg). The mean age was 6.5 years-old (range 5-9 years old).\u003c/p\u003e\n\u003cp\u003eUrine analysis \u0026ndash; The pH was between 4.5 and 8 with a urine specific gravity between 1.005-1.048. Discrete proteinuria was frequently and consistently observed in some individuals.\u003c/p\u003e\n\u003cp\u003eUrinary culture \u0026ndash; Urine cultures were mostly negative, with the exception of \u003cem\u003eStreptococcus infantarius\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e in estrus and \u003cem\u003eEnterococcus Hirae\u003c/em\u003e in diestrus in one individual and Lactobacillus Gasse in estrus in another.\u003c/p\u003e\n\u003cp\u003eVaginal microbiota \u0026ndash; A total of 315 genera were identified in the vaginal samples during the different cycle phases. \u0026nbsp;These most abundant bacterial populations belong to the genus \u003cem\u003eFusobacterium\u003c/em\u003e, \u003cem\u003ePorphyromas\u003c/em\u003e, \u003cem\u003eParvimonas\u003c/em\u003e and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e with a mean percentage of 33.1%, 11.5%, 7.1%, 7% and 5.8%, respectively in this global sample. To investigate the possible contaminant nature of each identified genus, a correlation test was applied between each identified genus and the bacterial abundance of the global population. The negative result (Rho -0,7972; p \u0026lt;0.0001) for \u003cem\u003eEscherichia-Shigella\u003c/em\u003e suggested that it was a contaminant. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicrobial population ecological indices of vaginal samples were assessed at the genus level. Results of alpha diversity, richness, and evenness of the bacterial populations throughout the different cycle phases were not significantly different (Figure 1).\u003c/p\u003e\n\u003cp\u003eGroup clustering testing did reveal significant overall differences between samples from the different cycle phases (Anestrus-Diestrus-Estrus-Proestrus) (p \u0026lt;0.0001) based on an AMOVA analysis. Paired analysis did reveal significant differences between anestrus and estrus (p \u0026lt;0.0001), anestrus and proestrus (p \u0026lt;0.0001), diestrus and estrus (p \u0026lt;0.0001), diestrus and proestrus (p \u0026lt;0.0001) and, estrus and proestrus (p \u0026lt;0.0002). No statistical difference was demonstrated between anestrus and diestrus. HOMOVA testing was statistically significant (p =0.0035). However, paired analysis did not yield statistically different results. Beta-diversity of vaginal microbial profile was visualized using an NMDS (non-metric dimensional scaling) model based upon a Bray-Curtis dissimilarity matrix including samples of the different cycle phases (Anestrus-Diestrus-Estrus-Proestrus), which is shown in Figure 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe AMOVA analysis resulted in statistical differences for beta diversity between the different cycle phases and paired difference analysis was significant except for anestrus and diestrus. Then differential population abundance between cycle phases was evaluated using a negative binomial Wald test as implemented in the DESeq2. No statistically significant differential population abundance between anestrus and diestrus was observed. There was a significant differential population abundance between proestrus and estrus for 5 genera (\u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eVariovorax\u003c/em\u003e and \u003cem\u003ePorphyromonas\u003c/em\u003e) comprising 2 contaminants. There was a significant differential population abundance between anestrus and estrus for 32 genera (\u003cem\u003eParvimonas, S5.A14, Peptostreptococcae genus, Anaerovoracacaea genus, Solobacterium, Porphyromonas, Fusobacterium, Alloprevotella, Peptococcus, Fusobacteriales genus, Porphyromonadacae genus, Johnsonella\u003c/em\u003e, \u0026hellip;) comprising 6 contaminants. There was a significant differential population abundance between anestrus and proestrus for 28 genera (\u003cem\u003eS5.A14a, Parvimonas, Porphyromonadacae genus, Anaerovoracaceae genus, Peptostreptococcaceae genus, Solobacterium, Alloprevotella,\u003c/em\u003e \u0026hellip;) comprising 7 contaminants. There was a significant differential population abundance between diestrus and proestrus for 27 genera (\u003cem\u003eS5.A14a, Porphyromonadacae, Fusobacterium\u003c/em\u003e, \u0026hellip;) comprising 10 contaminants. There was a significant differential population abundance between diestrus and estrus for 33 genera (\u003cem\u003eS5.A14a, porphyromonadacae, Fusobacterium, Bacteriodes, Peptostreptococcus, Johnsella, Parvimonas, Peptostreptococcaceae genus, Prophromonadaceae genius\u003c/em\u003e, \u0026hellip;) comprising 11 contaminants (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUrinary microbiota \u0026ndash; A total of 351 genera were identified in the urinary samples during the different cycle phases. \u0026nbsp;These most abundant bacterial populations belong to the genera \u003cem\u003eEscherichia-Shigella\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003eEnterobacteriaceae\u003c/em\u003e, \u003cem\u003ePseudomonadaceae\u003c/em\u003e and \u003cem\u003eRheinheimera\u003c/em\u003e with a mean percentage of 67%, 6.5%, 3%, 1.3% and 1%, respectively in this global sample. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGroup clustering testing didn\u0026rsquo;t reveal significant differences in variation between samples from the different cycle phases (Anestrus-Diestrus-Estrus-Proestrus) based on AMOVA analysis and HOMOVA testing yielded non-statistically significant differences (Figure 3).\u003c/p\u003e\n\u003cp\u003eMicrobial population ecological indices of vaginal samples were assessed at the genus level. Results of alpha diversity, richness and evenness of the bacterial populations throughout the different cycle phases were not significantly different (Figure 4).\u003c/p\u003e\n\u003cp\u003eNo statistical correlation was observed between urinary and vaginal microbiota.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe global composition of the vaginal microbiome includes bacteria, fungi, viruses, archaea, and candidate phyla radiation with bacteria communities representing the biggest percentage of the vaginal microbiome. The description of the different bacterial species can be interesting when looking for a particular species (i.e., pathogen) for example to study a pathology. In this study, we chose to describe the bacterial population according to the different genera encountered. This allowed precision while avoiding noise associated with identification by species. This best suited the purpose of our research, which was to characterize and compare the urogenital microbiota in the different phases of the estrous cycle in healthy bitches.\u003c/p\u003e \u003cp\u003eEarly studies characterizing vaginal microbiota by aerobic and anaerobic culture methods in bitches yielded \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. pseudintermedius\u003c/em\u003e as the most common isolates.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e It has been suspected to be a limitation of routine culture. With the 16S rDNA gene sequencing, \u003cem\u003eHydrotalea, Ralstonia, Mycoplasma, Fusobacterium\u003c/em\u003e and \u003cem\u003eStreptoccocus\u003c/em\u003e were reported to be the predominant genera in the vagina of female dogs in the study of Burton et al. 2017.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eMycoplasma, Pasteurellaceae\u003c/em\u003e family and \u003cem\u003eSalmonella\u003c/em\u003e were identified in healthy bitches of various breed in the study of Rota et al. 2020\u003csup\u003e20\u003c/sup\u003e and \u003cem\u003eFusobacteria, Firmicutes, Proteobacteria, Tenericutes\u003c/em\u003e and \u003cem\u003eBacteroidetes\u003c/em\u003e were identified in beagles in the study of Hu et al. 2022.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The results of our study partly agree with the studies of Burton et al.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e and Hu et al.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e In our population, \u003cem\u003eFusobacterium, Porphyromas\u003c/em\u003e, \u003cem\u003eParvimonas\u003c/em\u003e and \u003cem\u003eEscherichia-Shigella\u003c/em\u003e were the predominant genera. As \u003cem\u003eEscherichia-Shigella\u003c/em\u003e was suspected to be a contaminant, like in the evaluation by bacterial culture, the remaining predominant genera in our study are \u003cem\u003eFusobacterium, Porphyromas\u003c/em\u003e and \u003cem\u003eParvimonas\u003c/em\u003e. In cyclic women, vaginal microbiota is mostly dominated by \u003cem\u003eLactobacillus crispatus, L. iners, L. gasseri\u003c/em\u003e or \u003cem\u003eL. jensenii\u003c/em\u003e contrary to the observations of our study.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEarly studies using aerobic and anaerobic culture characterized urine as sterile.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e With the 16S rDNA gene sequencing, \u003cem\u003ePhylum Pseudomonadota\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e sp, \u003cem\u003eAcinetobacter\u003c/em\u003e sp, \u003cem\u003eSphingobium\u003c/em\u003e sp and \u003cem\u003eBradyrhizobiaceae\u003c/em\u003e were reported to dominate the urinary microbiota in dogs in a previous study.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The results of our study were partly in accordance with those of the previous report. In our population, \u003cem\u003eEscherichia-Shigella\u003c/em\u003e, \u003cem\u003eFlavobacterium\u003c/em\u003e, \u003cem\u003eEnterobacteriaceae genus\u003c/em\u003e, \u003cem\u003ePseudomonadaceae genus\u003c/em\u003e and \u003cem\u003eRheinheimera\u003c/em\u003e were the dominant genera. \u003cem\u003ePseudomonas\u003c/em\u003e sp is part of the \u003cem\u003ePseudomonadaceae\u003c/em\u003e family. Moreover, \u003cem\u003eLactobacillus Gasse\u003c/em\u003e was identified in urine at the culture and confirmed by the 16S rDNA gene sequencing in one dog but was not identified in her vaginal microbiota. \u003cem\u003eEscherichia-Shigella, Enterobacteriaceae\u003c/em\u003e, \u003cem\u003ePseudomonadaceae\u003c/em\u003e and \u003cem\u003eRheinheimera\u003c/em\u003e are parts of the \u003cem\u003ePseudomonadota\u003c/em\u003e phylum. \u003cem\u003eSphingobium\u003c/em\u003e, \u003cem\u003eAcinetobacter, Rheinheimera and Flavobacterium\u003c/em\u003e are bacteria that have been observed in water. \u003cem\u003eCorynebacterium, Streptococcus\u003c/em\u003e and \u003cem\u003eActinomyces\u003c/em\u003e, reported as part of women urinary microbiota, are found in aqueous media, but not exclusively.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e None of the bacteria of a woman\u0026rsquo;s urinary microbiota is therefore specific to the aqueous environment.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e This difference between the urinary microbiota of women and bitches is currently unexplained. It can be speculated that anatomical differences may account for the discrepancy or that urinary microbiota could be different depending on the species, as reported for the intestinal microbiota.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTo identify potential contaminant, a correlation test between the presence of the genus and the abundance of the bacterial population of the vagina was performed. \u003cem\u003eEscherichia-Shigella, Comamonadaceae genus, Pseudomanas, Flavobacteriaceae genus, Enterobacterales genus, Flavobacterium, Rheinheimera, Pelomonas, Acinetobacter, saccharimonadales genus, Chryseobacterium genus, Parcubactera genus, Aeromonas, Burkholderiales genus and Paracoccus\u003c/em\u003e should be considered as contaminants. Interestingly, \u003cem\u003eEscherichia-Shigella, Flavobacterium\u003c/em\u003e, \u003cem\u003eEnterobacteriaceae genus\u003c/em\u003e and \u003cem\u003eRheinheimera\u003c/em\u003e, which were dominant genera in urinary microbiota, are also found in the vaginal microbiota. We supposed that this is more an overlap of both microbiota than a real contamination. In a previous study, the genital microbiome was similar to the urinary microbiota.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e However, a correlation test let suspect that \u003cem\u003eEscherichia-Shigella, Flavobacterium\u003c/em\u003e, \u003cem\u003eEnterobacteriaceae genus\u003c/em\u003e and \u003cem\u003eRheinheimera\u003c/em\u003e were vaginal contaminants and no correlation between vaginal and urinary microbiota was statistically demonstrated in our study. Thus, a contamination of the vaginal microbiota by urine during sampling can\u0026rsquo;t be excluded. A previous study showed similar microbiota in dogs with or without recurrent urinary tract infection, which seems to discard the condition as a source of variation in the vaginal microbiota.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Also, contaminants may come from the animal or from the environment or food during the collection procedure. In reports, in humans, an influence of the food on the urinary and vaginal microbiota has been described.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Our dog population is made up of laboratory beagles fed a strictly normal or light dry food diet, as opposed to a population of healthy dogs from owners who will tend to have a more varied diet including dry food/wet food as well as table scraps. They live in a kennel with wood shavings as bedding, which is cleaned daily and changed weekly, which is very different from the classic environment of owner dogs living indoors or in outdoor parks. Therefore, the external environment in which the dogs\u0026rsquo; lives could be more contaminated (e.g., through litter with wood chips or feces) than the conventional environment of a pet. Although urine and vaginal samples were collected in a sterile environment, contamination cannot be definitively ruled out. Urogenital microbiota analysis of a group of female dogs living in a conventional environment and comparison with those of the beagles living in a laboratory kennel would be necessary to confirm this hypothesis.\u003c/p\u003e \u003cp\u003eSex hormones have been demonstrated to contribute to the regulation of vaginal microbiota \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, as it covaries with estradiol level and differs in alpha and beta diversity across the menstrual cycle of women.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The influence of progesterone is not well known. The results of our study showed a variation of the vaginal microbiota according to the cycle of the bitches. Alpha diversity, which corresponds to the number of species coexisting in the vagina, was not statistically different during the cycle. Absence of influence on alpha diversity in our study compared with the study of Song et al. 2020\u003csup\u003e25\u003c/sup\u003e may be due to the fact that we analyzed the microbiota once per cyclic phase compared with this study that did it once daily or can be due to the difference of species between dogs and women. No influence of progesterone on vaginal microbiota could be shown in our study as beta diversity did not differ between diestrus, when progesterone is highest, and anestrus, when there are no significant levels of circulating sex hormones. Indeed, estradiol-β17 concentration peaks during proestrus and decreases during estrus to be at the lowest concentration during diestrus and anestrus. In contrast, the progesterone concentration increases during the estrus to be at the highest concentration during diestrus. Progesterone concentration is low during anestrus and proestrus.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Therefore, the variation of beta diversity during estrogenic phases compared to diestrus and anestrus highlights the influence of estrogens on the vaginal microbiome as described in woman.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn women, estrogens induce a stimulation of vaginal epithelial cells proliferation, with a mid-cycle peak in intracellular glycogen levels in the vaginal mucosa and a subsequent increase in lactic acid producing microbes, such as \u003cem\u003eLactobacillus\u003c/em\u003e, in the vaginal milieu.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e While similar effect of estrogens on vaginal cells proliferation exists in the dog\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, the mechanism explaining the changes in the microbiome remain to be explored. Moreover, \u003cem\u003eLactobacillus\u003c/em\u003e hasn\u0026rsquo;t been identified in vaginal microbiota in our population of middle-aged females what suggests a really different vaginal environment between women and dogs contrary to the study of Hu J. et al. 2022 in which, \u003cem\u003eLactobacillus\u003c/em\u003e was present and increased in proportion as the dogs aged.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The difference between the vagina in bitches and women, with a pH of 7 and 4.5 respectively, could explain why the microbiota composition is significantly different.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Other factors that could influence vaginal microbiota during the cycle include the presence of blood in the environment during proestrus, which may contribute to the changes in beta-diversity by the presence of iron or increased pH.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e The blood could also influence the vaginal microbiota by providing substrate for growth proliferation or flushing out bacteria.\u003c/p\u003e \u003cp\u003eWe postulated that estrogens could influence the urinary microbiota in bitches because urodynamics studies have shown an influence of the estrous cycle and the sex hormones on urinary system by decreasing urethral pressure during estrous and early diestrus.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Estrogens induce an increase in the number of alpha-adrenergic receptors and responsiveness of these receptors to sympathetic stimulation.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Estrogens also induce an increase in blood flow to urethral tissues\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, which causes an increase in urethral sphincter tone.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Progesterone potentiates beta-adrenergic activity in urethra of female dogs, leading to relaxation and a decrease in urethral smooth muscle tone.\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e However, no influence of the cycle and thus of the estrogenic impregnation on alpha and beta diversity of the urinary microbiota could be demonstrated in this study. The absence of variation of alpha and beta diversity of the urinary microbiota may be due to the absence of change in the environment during the cycle. Indeed, if estrogens and progesterone have an indirect action on urethral pressure, there is no reported change in the urinary system structure, namely the epithelium, unlike for the vagina.\u003c/p\u003e \u003cp\u003eThe main limitations in this study were the possible presence of contaminants in the sample despite all the aseptic methods used for collection and the small number of dogs included. Another source of contamination could come from bedding in the kennel even if the litter was changed weekly and cleaned daily. However, this was a homogenous group of dogs regarding the breed, age, housing conditions and food.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe perspectives of this study are numerous. The next step will be to describe the evolution of the urinary and genital microbiota in growing healthy male and female dogs, and afterwards to study the urogenital microbiota in pathological conditions.\u003c/p\u003e \u003cp\u003eTo conclude, this study demonstrated an estrogenic influence on the abundance of vaginal microbiota in healthy female dogs and provided a comparative basis of urinary and vaginal microbiota in healthy female dogs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003ea. Ethics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThis research had the ULiège Ethical agreement number: 20-2250\u003c/p\u003e\n\u003cp\u003eb. Consent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable. Participants are laboratory dogs for which participation has been authorized by the ULiège Ethical agreement number: 20-2250\u003c/p\u003e\n\u003cp\u003ec. Availability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data of sample and statistical analysis are available on request to Virginie Gronsfeld\u003c/p\u003e\n\u003cp\u003ed. Competing interests\u003c/p\u003e\n\u003cp\u003eThe authors report there are no competing interests to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ee. Author contributions:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eV.G. Wrote the Ian manuscript text\u003c/p\u003e\n\u003cp\u003eF.B., S.E., C.P. Participated to the collection of data\u003c/p\u003e\n\u003cp\u003eA.H. and G.D. Provided material\u003c/p\u003e\n\u003cp\u003eB.T. Did the analysis of data\u003c/p\u003e\n\u003cp\u003eM-L.VdW. Did the analysis of sample\u003c/p\u003e\n\u003cp\u003eS.D. and S.N. reviewed the manuscript\u003c/p\u003e\n\u003cp\u003ef. Funding\u003c/p\u003e\n\u003cp\u003eThe authors have no funding to declare.\u003c/p\u003e\n\u003cp\u003eg. Acknowledgments\u003c/p\u003e\n\u003cp\u003e/\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTheron J, Cloete TE. Molecular techniques for determining microbial diversity and community structure in natural environments. Crit Rev Microbiol 2000; 26(1):37‐57.https://doi.org/10.1080/10408410091154174\u003c/li\u003e\n\u003cli\u003eCoffey EL, Gomez AM, Ericsson AC, Burton EN, Granick JL, Lulich JP et al. The impact of urine collection method on canine urinary microbiota detection: a cross-sectional study. BMC Microbiol 2023; 23(1):101https//doi.org/10.1186/s12866-023-02815-y\u003c/li\u003e\n\u003cli\u003eHilt EE, McKinley K, Pearce MM, Rosenfeld AB, Zilliox MJ, Mueller ER et al. Urine is not sterile: use of enhanced urine culture techniques to detect resident bacterial flora in the adult female bladder. J Clin Microbial 2014; 52:871‐876. https://doi.org/10.1128/JCM.02876-13\u003c/li\u003e\n\u003cli\u003ePearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, Fok C, et al. The female urinary microbiome: a comparison of women with and without urgency urinary incontinence. mBio 2014; e01283\u0026ndash;14.https://doi.org/10.1128/mBio.01283-14\u003c/li\u003e\n\u003cli\u003eThomas-White K, Forster SC, Kumar N, Van Kuiken M, Putonti C, Stares MD et al. Culturing of female bladder bacteria reveals an interconnected urogenital microbiota. Nat commun 2018; 9(1):1557https://doi.org/10.1038/s41467-018-03968-5\u003c/li\u003e\n\u003cli\u003eShen J, Song N, Williams CJ, Brown CJ, Yan Z, Xu C et al. Effects of low dose estrogen therapy on the vaginal microbiomes of women with atrophic vaginitis. Sci Rep 2016;29(6):34119.https://doi.org/10.1038/srep34119\u003c/li\u003e\n\u003cli\u003eChen KL, Madak-Erdogan Z. Estrogen and Microbiota Crosstalk: Should We Pay Attention? Trends Endocrinol Metab 2016;27(11):752‐755.https://doi.org/10.1016/j.tem.2016.08.001\u003c/li\u003e\n\u003cli\u003eRomero R, Hassan SS, Gajer P, Tarca AL, Fadrosh DW, Nikita L et al. The composition and stability of the vaginal microbiota of normal pregnant women is different from that of non-pregnant women. Microbiome 2014;2(1):4. https://doi.org/10.1186/2049-2618-2-4\u003c/li\u003e\n\u003cli\u003eKaur H, Merchant M, Haque MM, Mande SS. Crosstalk Between Female Gonadal Hormones and Vaginal Microbiota Across Various Phases of Women\u0026apos;s Gynecological Lifecycle. Front Microbiol 2020;11:551. https://doi.org/10.3389/fmicb.2020.00551\u003c/li\u003e\n\u003cli\u003eSmith SB, Ravel J. The vaginal microbiota, host defence and reproductive physiology. J Physiol 2017;595(2):451‐463. https://doi.org/10.1113/JP271694\u003c/li\u003e\n\u003cli\u003eChaban B, Links MG, Jayaprakash TP, Wagner EC, Bourque DK, Lohn Z et al. Characterization of the vaginal microbiota of healthy Canadian women through the menstrual cycle. Microbiome 2014;2:23. https://doi.org/10.1186/2049-2618-2-23\u003c/li\u003e\n\u003cli\u003eBurton EN, Cohn LA, Reinero CN, Rindt H, Moore SG, Ericsson A. Characterization of the urinary microbiome in healthy dogs. PloS One 2017;12(5):e0177783. https//doi.org/10.1371/journal.pone.0177783 \u003c/li\u003e\n\u003cli\u003eLyman CC, Holyoak GR, Meinkoth K, Wieneke X, Chillemi KA, DeSilva U. Canine endometrial and vaginal microbiomes reveal distinct and complex ecosystems. PLoS One 2019;14(1):e0210157. https//doi.org/10.1371/journal.pone.0210157\u003c/li\u003e\n\u003cli\u003eGolinskan E, Sowinska N, Tomusiak-Plebanek A, Szydlo M, Witka N, Lenarczyk J et al. The vaginal microflora changes in various stages of the estrous cycle of healthy female dogs and the ones with genital tract infections. BMC Vet Res, 17(1):8. https//doi.org/10.1186/s12917-020-02710-y\u003c/li\u003e\n\u003cli\u003eHu J, Cui L, Wang X, Gao X, Qiu S, Qi H et al. Dynamics of vaginal microbiome in female beagles at different ages. Res Vet Sci 2022; 149:128\u0026ndash;135. https//doi.org/10.1016/j.rvsc.2022.05.006\u003c/li\u003e\n\u003cli\u003eHamaide AJ, Verstegen JP, Snaps FR, Onclin KJ, Balligand MH. Influence of the estrous cycle on urodynamic and morphometric measurements of the lower portion of the urogenital tract in dogs. Am J Vet Res 2005;66(6):1075\u0026ndash;1083. https//doi.org/10.2460/ajvr.2005.66.1075\u003c/li\u003e\n\u003cli\u003eMrofchak R, Madden C, Evans MV et Hale VL. Evaluating extraction methods to study canine urine microbiota. PLoS One 2021;16(7):e0253989 https//doi.org/10.1371/journal.pone.0253989\u003c/li\u003e\n\u003cli\u003eGupta S, Mortensen MS, Schj\u0026oslash;rring S, Trivedi U, Vestergaard G, Stokholm J et al. Amplicon sequencing provides more accurate microbiome information in healthy children compared to culturing; Commun Biol 2019;2:291. https//doi.org/10.1038/s42003-019-0540-1\u003c/li\u003e\n\u003cli\u003eHutchins RG, Vaden SL, Jacob ME, Bowles KD, Wood MW, Bailey CS et al. Vaginal microbiota of Spayed dogs with or without recurrent urinary tract infections. J Vet Intern Med 2014, 28: 300-304. https//doi.org/10.1111/jvim.12299\u003c/li\u003e\n\u003cli\u003eRota A, Corr\u0026ograve; M, Patuzzi I, Milani C, Masia S, Mastrorilli E et al. Effect of sterilization on the canine vaginal microbiota: a pilot study. BMC Vet Res 2020;16(1):455. https//doi.org/10.1186/s12917-020-02670-3\u003c/li\u003e\n\u003cli\u003eRavel J, Gajer P, Abdo Z, et al. Vaginal microbiome of reproductive-age women. Proc Natl Acad Sci, 2011; 108:4680‐7. https://doi.org/10.1073/pnas.1002611107.\u003c/li\u003e\n\u003cli\u003eWolfe AJ, Toh E, Shibata N, Rong R, Kenton K, et al. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol. 2012;50:1376\u0026ndash;1383. https://doi.org/10.1128/jcm.02876-13\u003c/li\u003e\n\u003cli\u003eDeng P and Swanson KS. Gut microbiota of humans, dogs and cats: current knowledge and future opportunities and challenges. Br J Nutr 2015;113 Suppl:S6-17. https//doi.org/10.1017/S0007114514002943\u003c/li\u003e\n\u003cli\u003eFengping L, Zongxin L, Chulei T, Yi F, Chen YQ et al. Moderation effects of food intake on the relationship between urinary microbiota and urinary interleukin-8 in female type 2 diabetic patients. PeerJ 2020;8: e8481. https//doi.org/10.7717/peerj.8481\u003c/li\u003e\n\u003cli\u003eSong SD, Acharya KD, Zhu JE. Daily Vaginal Microbiota Fluctuations Associated with Natural Hormonal Cycle, Contraceptives, Diet, and Exercise. mSphere 2020; 5(4):e00593-20. https//doi.org/10.1128/mSphere.00593-20\u003c/li\u003e\n\u003cli\u003eOlson PN, Bowen RA, Behrendt MD, Olson JD, Nett TM. Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus and estrus. Biol Reprod 1982, 27(5):1196-206. https//doi.org/10.1095/biolreprod27.5.1196\u003c/li\u003e\n\u003cli\u003eFarage MA, Miller KW, and Sobe JD. Dynamics of the Vaginal Ecosystem\u0026mdash;Hormonal Influences. Infectious Diseases: Research and Treatment 2010; 3:1-15. https//doi.org/10.4137/IDRT.S3903\u003c/li\u003e\n\u003cli\u003eJohnston SD, Root Kustritz MV, Olson PN: Vaginal cytology. In: Kersey R, LeMelledo D, eds. Canine and feline theriogenology. Philadelphia, PA: Saunders; 2001, pp. 32\u0026ndash;40.\u003c/li\u003e\n\u003cli\u003eNoël SM, Farnir F, Hamaide AJ. Urodynamic and morphometric characteristics of the lower urogenital tracts of female Beagle littermates during the sexually immature period and first and second estrous cycles . Am J Vet Res 2012;73:1657-1664. https//doi.org/10.2460/ajvr.73.10.1657\u003c/li\u003e\n\u003cli\u003eRaz S, Caine M, Zeigler M. The vascular component in the production of intraurethral pressure. J Urol 1972;108(1):93‐96. https//doi.org/10.1016/s0022-5347(17)60650-5\u003c/li\u003e\n\u003cli\u003eRaz S, Zeigler M, Caine M. Br. The effect of progesterone on the adrenergic receptors of the urethra. J Urol 1972;45(2):131‐5. https//doi.org/10.1111/j.1464-410x.1973.tb12129.x\u003c/li\u003e\n\u003cli\u003eCaine M, Raz S. Some clinical implications of adrenergic receptors in the urinary tract. Arch Surg 1975;110(3):247‐50. https//doi.org/10.1001/archsurg.1975.01360090017003\u003c/li\u003e\n\u003cli\u003eHolt PE. Urinary incontinence in the bitch due to sphincter mechanism incompetence: prevalence in referred dogs and retrospective analysis of sixty cases. 1985, Small Anim Pract, 26:181\u0026ndash;190. https://doi.org/10.1111/j.1748-5827.1985.tb02099.x\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Vaginal microbiota, urinary microbiota, hormonal influence, dogs","lastPublishedDoi":"10.21203/rs.3.rs-3955899/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3955899/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Understanding the urogenital microbiota would allow us to compare the bacterial populations in healthy and pathological conditions and assess their impact on various urogenital diseases. The aim of our research was to characterize and compare the urogenital microbiota during different phases of the estrous cycle in healthy female dogs. DNA extraction, 16S rDNA library preparation, sequencing, and informatics analysis were employed to determine the vaginal and urinary microbiota in 10 healthy beagle dogs at each phase of the cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Alpha diversity, richness, and evenness of bacterial populations in the vagina were not significantly different across the various cycle phases. However, there was a significant difference in vaginal beta diversity between the different cycle phases, except for anestrus and diestrus. Conversely, no differences in alpha and beta diversity were observed in the urinary microbiota across the different cycle phases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study demonstrate estrogenic influence on the abundance of vaginal microbiota in healthy female dogs, with no discernible influence on urinary microbiota. Additionally, it provides a comparative basis for understanding the urinary and vaginal microbiota in healthy female dogs.\u003c/p\u003e","manuscriptTitle":"Evaluation of the urogenital microbiota of healthy cyclic bitches","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-19 13:12:36","doi":"10.21203/rs.3.rs-3955899/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-20T14:55:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-15T16:29:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-15T12:02:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Veterinary Research","date":"2024-02-14T10:57:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-veterinary-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [BMC Veterinary Research](http://bmcvetres.biomedcentral.com/)","snPcode":"12917","submissionUrl":"https://submission.nature.com/new-submission/12917/3?","title":"BMC Veterinary Research","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"16fa58bc-b093-42ef-adaf-eb80366a8079","owner":[],"postedDate":"February 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:15:17+00:00","versionOfRecord":{"articleIdentity":"rs-3955899","link":"https://doi.org/10.1186/s12917-024-04104-w","journal":{"identity":"bmc-veterinary-research","isVorOnly":false,"title":"BMC Veterinary Research"},"publishedOn":"2024-07-15 16:13:41","publishedOnDateReadable":"July 15th, 2024"},"versionCreatedAt":"2024-02-19 13:12:36","video":"","vorDoi":"10.1186/s12917-024-04104-w","vorDoiUrl":"https://doi.org/10.1186/s12917-024-04104-w","workflowStages":[]},"version":"v1","identity":"rs-3955899","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3955899","identity":"rs-3955899","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}