Expression pattern of germ cell markers in cryptorchid stallion testes.

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions.

The reproductive microbiome in dogs: Friend or foe?

The microbiome of the reproductive tract is an area of research in full development. Specifically, the microbiome may be involved in reproductive health, disease, and pregnancy outcomes, as has been shown in humans and animals, including dogs. The aim of the present review was to summarize current knowledge on the microbiome of the canine reproductive tract, to expose the controversial role that some bacterial agents may play in canine subfertility, and to highlight future research perspectives. This review discussed whether the use of antimicrobials in dogs is appropriate to increase reproductive performance and to treat subfertility without proper diagnosis, and the possible use of probiotics to modulate the reproductive canine microbiome. Finally, we indicate areas in which scientific knowledge is currently lacking, and could be promising directions for future research.

Bicavitary effusion in cats: retrospective analysis of signalment, clinical investigations, diagnosis and outcome.

OBJECTIVES: The aim of this study was to describe the clinical and diagnostic findings and outcome of cats with bicavitary effusion presenting to a referral centre. METHODS: Medical records of cats presenting with bicavitary effusion were identified and their history, physical examination findings, clinicopathological data, diagnostic imaging findings, aetiology of bicavitary effusions (cardiac disease, neoplasia, infectious disease, sterile inflammatory disease, severe hypoalbuminaemia, trauma, coagulopathy or 'open' if no definitive diagnosis was reached) and outcome were recorded. Cox regression analysis was performed to identify independent predictors of death in cats with bicavitary effusion. Kaplan-Meier curves were generated for survival analysis. RESULTS: In total, 103 cats with bicavitary effusion were included. Neoplasia and cardiac disease were the most common aetiologies of bicavitary effusion, in 21 (20.4%) and 20 (19.4%) cats, respectively, followed by infectious disease (n = 11, 10.7%), trauma (n = 13, 12.6%), hypoalbuminaemia (n = 6, 5.8%), sterile inflammatory disease (n = 4, 3.9%) and coagulopathy (n = 1, 1.0%). The median survival time for all cats with bicavitary effusion was 3 days. Cats with a neoplastic aetiology had a 2.03 times greater risk of death compared with cats in which no diagnosis was achieved. Neoplasia (P = 0.030) and pedigree breed status (P = 0.016) were independent predictors of death in the multivariable Cox regression model. CONCLUSIONS AND RELEVANCE: This study highlights that bicavitary effusions in cats generally carry a guarded to poor prognosis, particularly if neoplasia is the underlying aetiology or if the cat is a pedigree breed. Cardiac disease appeared to be associated with a better prognosis, suggesting that assessment for congestive heart failure should be considered early when evaluating cats with bicavitary effusion. The prognosis for cats with feline infectious peritonitis is likely to be markedly improved by the advent of novel antiviral drugs, compared with the historical cohort of cats presented here.

Development of alternative diagnosis of HH1, HH3, HH5 and HCD fertility haplotypes and subfertility syndrome in cattle.

The increasing prevalence of hereditary anomalies in Holstein cattle populations presents a pressing issue, leading to concerns such as embryonic mortality and the birth of non-viable offspring. This study addresses the urgency of managing harmful genetic mutations in Holstein cattle by developing alternative diagnostic methods. The research aims to devise effective means to diagnose fertility haplotypes HH1, HH3, HH5, HCD and BY and subfertility syndrome in cattle. To achieve this goal, a range of molecular genetic techniques were employed, including Tetra-Primer ARMS-PCR methods, PCR-RFLP analysis and allele-specific PCR. These methods facilitated the identification of heterozygous carriers of various fertility haplotypes and subfertility syndrome in Holstein cows and servicing bulls. The study reveals the prevalence of these genetic defects within the Republic of Kazakhstan's cattle population. HH1, HH3, HH5, HCD and BY fertility haplotypes were found to have occurrence rates ranging from 1.4% to 16.6%, with subfertility syndrome detected in 4.5% of Simmental bulls. The practical significance of this research lies in its contribution to genetic monitoring and management strategies for Holstein cattle populations. By introducing affordable, rapid and accurate diagnostic methods, such as the T-ARMS-PCR, the study provides a valuable tool for controlling and mitigating the spread of harmful genetic mutations, ultimately improving the overall genetic health and productivity of Holstein cattle in the region. This research addresses a critical need in the cattle breeding industry and underscores the importance of genetic monitoring to ensure the long-term viability and sustainability of Holstein cattle populations.

Ancillary Methods for Semen Evaluation.

The cause of subfertility or poor fertility in naturally mated bulls should be differentiated from impotentia coeundi, generandi, or erigendi prior to ancillary semen evaluation. Bulls used for artificial insemination may undergo ancillary semen evaluation following low fertility rates as judged by poor conception or low pregnancy rates. Morphologically abnormal sperm have long been associated with bull subfertility and infertility. Some morphological defects such as improper sperm chromatin condensation are not visible using traditional light microscopy and require specialized staining. Ancillary semen evaluation is useful in cases where the reason for low or absence of fertility needs to be identified. As compared to SEM, TEM can be extremely useful for identifying minuscule acrosome defects, issues with chromatin, and centrosome defects and is considered the gold standard method for the identification of midpiece and tail defects.

Medical and Surgical Management of Conditions of the Penis and Prepuce.

The inability of a bull to reproduce due to its inability to impregnant fertile cows is called impotentia generandi. This infertility may be due to the inability to achieve erection, the inability to complete coitus, or the inability to produce an adequate volume of morphologically normal spermatozoa. Therapies targeting the urogenital tract of the bull can restore reproductive capabilities. Veterinarians can provide consultation regarding both management and selection criteria that will, in some cases, lower the overall risk of loss associated with the development of some conditions of the penis and prepuce.

Abnormalities of the Scrotum and Testes.

Abnormalities of the bovine scrotum and testes are an important cause of infertility. Proper evaluation of the male reproductive system is a critical first step in screening for such abnormalities. Excessive periscrotal fat, cutaneous scrotal defects, and unilateral scrotal swelling are common deformities that warrant further investigation. Early diagnosis and surgical intervention are often needed to restore reproductive soundness. This article reviews these conditions and provides therapeutic modalities.

Is glycerol a good cryoprotectant for sperm cells? New exploration of its toxicity using avian model.

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation.

Sperm abnormality and infertility in male mice treated with the recommended dose of dimethoate and its double.

Background: Dimethoate (DM) is one of the most important organophosphate insecticides used for controlling many pests which affect vegetables, fruits, and agricultural crops, its persistence in soils and crops could cause a health hazard to humans as well as other non-target organisms. Aim: This study was conducted to evaluate the effect of the recommended dose and its double of DM on sex hormones, sperm morphology, and fertility of adult male mice. Methods: Twenty-seven Swiss albino adult male mice were divided into three groups of nine animals each: control group received distilled water only, while other groups received DM orally at doses (0.1 and 0.2 ml DM/100 ml distilled water) for 20 days, at the end of the treatment, six mice from each group were sacrificed. The sperm morphology was evaluated and sex hormones were measured. Three mice from each group were allowed to mate with untreated females (1:2). Result: The results revealed a decrease in luteinizing hormone levels in mice treated with (0.2 ml DM/100 ml distilled water) compared with the control group while the levels of follicle-stimulating hormone and testosterone did not record any significant differences. Also, the results demonstrated a significant increase in abnormal sperm morphology such as head and tail. The fertility was reduced and the average number of dead embryos increased while the average number of live embryos decreased. Conclusion: This current study confirmed that DM has detrimental effects on sperm morphology, fertility, and the embryos; therefore, more efforts should be exerted to protect ourselves and our environment from the harmful effects of this pesticide.

Refrigerated multi-dose insulin vials remain sterile through 6 months of use.

OBJECTIVES: To evaluate sterility in refrigerated multi-dose insulin vials through 6 months of routine aspiration. MATERIALS AND METHODS: Twelve vials of insulin, six of insulin glargine U100 (Lantus®, 10 mL multi-dose vial, Sanofi, Bridgewater, NJ) containing the preservative metacresol, and six of protamine zinc insulin U40 (ProZinc®, 10 mL multi-dose vial, Boehringer Ingelheim, Duluth, GA) containing the preservative phenol, were refrigerated and aspirated twice daily for 6 months, using a new insulin syringe each time. Three vials of each insulin type were wiped with a single-use alcohol swab before sampling. Three times weekly, aspirated samples were inoculated in Tryptic Soy Broth enrichment media and incubated for evidence of microbial growth. Positive broth was cultured and speciated. Endpoints were microbial vial contamination (defined as three consecutive positive cultures of the same organism) and completion of the six-month study period. RESULTS: Microbial contamination was not identified in any vial throughout the study period. A total of 454 aspirated samples were cultured, one of which exhibited non-repeatable growth of Staphylococcus epidermidis. This vial was prematurely lost to breakage after 59 culture samples (29 after the positive growth). CLINICAL SIGNIFICANCE: Refrigerated phenol- and metacresol-containing multi-dose insulin products carry minimal risk for iatrogenic infection through 6 months of use, regardless of alcohol swab preparation.

Causes of pregnancy arrest in the canine species.

In the canine species, early pregnancy arrest before 30-40 days of pregnancy will induce intra-uterine embryonic or foetal resorption, with very few clinical signs. If no genital examination by ultrasound is performed at that time, it will often remain unnoticed and the bitch will be qualified as infertile. It is only when pregnancy stops at a later stage, mostly after 40 days, that clinical signs will be seen. Expulsion of aborted foetuses or placentas may be observed, although the dam frequently eats the expelled tissues. Intra-uterine mummification may also occur. This article reviews the causes of pregnancy termination in the bitch, both at the embryonic and the foetal stages, reported in the literature. Canine brucellosis is by far the most important disease concerned in this aspect. There is an important current concern about this disease, due to the fact that several outbreaks have been observed recently in Europe, and that it is a very contagious disease; it may be an underestimated zoonosis. Other bacterial causes of pregnancy arrest are sporadic. There is, however, a growing attention towards the microbiological content of raw food diets which are more and more popular among dog breeders and may-if badly prepared-contain abortifacient bacteria such as Campylobacter jejuni or Listeria monocytogenes. The abortifacient role of endogenous vaginal bacteria and mycoplasms remains unclear and may be related to an imbalanced vaginal flora with subsequent ascending bacterial contamination of the uterus. The role of Canine Herpesvirus on abortion is controversial and probably not frequent. Other viruses have been shown experimentally to induce abortion but their natural occurrence in this respect remains unknown. The same applies to the parasite Neospora caninum which is suspected, but not proven, potentially to induce pregnancy arrest in bitches. Among non-infectious causes, uterine pathology such as cystic endometrial hyperplasia (CEH) or sub-clinical post-mating endometritis which can cause infertility and may also induce embryonic resorption. The role of luteal insufficiency in pregnancy arrest is probably overestimated.

Detection of Some Causes of Feline Eye Infections in Baghdad City.

The current study aimed to determine the causes associated with ocular infection in cats received at Baghdad veterinary hospital from March 2020 to April 2021. Forty cats (22 females and 18 males) were examined at a small animal clinic in Baghdad veterinary hospital from March 2020 to April 2021. The cats suffered from severe eyes infection (inflammation, lacrimation, redness and other ocular signs). On the other hand, ten healthy cats were examined and prepared for bacterial isolation as a control group. For bacterial isolation, sterile cotton swabs with transport medium were taken gently from the corneal and conjunctiva area of infected eyes. The swabs were placed in an ice box within 24 hours for laboratory culture. Sterile swabs with transport media were used in our study; swabs passed directly on the inferior conjunctival sac of the compromised eye avoiding contact with eyelashes and skin of eyelids. All swabs were inoculated on the following media (5% Sheep blood agar, MacConkey agar and Nutrient agar) at 37ºC for 24 to 48 h.ImmunoChromatoGraphy assay (ICG) of FCV on samples. The results showed that 50%of Mixed bacterial and FCV were the significant cause of isolates; also, it showed that S. aureus was the most bacterial cause of eye infection; young females were mostly infected in February. In conclusion, the wide distribution of ocular infections in cats is due to different causes, especially with bacteria, including Staphylococcus spp. and virus (FCV). The seasonal variation between months plays a significant factor in the spreading of eye infections in the feline.

Characterization and identification of extracellular vesicles-coupled miRNA profiles in seminal plasma of fertile and subfertile rabbit bucks.

Seminal plasma (SP) provides essential nutrients, transport, and protection to the spermatozoa during their journey through the male and female reproductive tracts. Extracellular vesicles (EVs) are one of the main components of the SP with several biomolecular cargoes, including miRNAs, that can influence spermatozoa functions and interact with the cells of the female reproductive tract. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SP-EVs isolated from fertile (F) and subfertile (S) rabbit bucks that could serve as fertility biomarkers. In this study, the methods to isolate and identify EVs including exosomes, from SP of 3 F and S bucks have been developed. Ultracentrifugation and size exclusion chromatography analysis were using to isolate EVs from SP of F and S males that were qualitative and quantitively characterised using transmission electron microscopy, nanoparticle tracking analysis and western blotting. In addition, total RNA, including miRNA, was isolated, sequenced and identified from SP-EVs samples. Different SP-EVs concentrations (8.53 × 1011 ± 1.04 × 1011 and 1.84 × 1012 ± 1.75 × 1011 particles/mL of SP; P = 0.008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; P = 0.7422) in F and S males, respectively was observed. Particle size was not significantly correlated with any kinetic parameter. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r = 0.94; P < 0.05) and with the percentage of immotile spermatozoa (r = 0.88; P < 0.05). Small-RNA-seq analysis identified a total of 267 and 244 expressed miRNAs in the F and S groups, respectively. Two miRNAs (let-7b-5p and let-7a-5p) were the top most abundant miRNAs in both groups. Differential expression analysis revealed that 9 miRNAs including miR-190b-5p, miR-193b-5p, let-7b-3p, and miR-378-3p, and another 9 miRNAs including miR-7a-5p, miR-33a-5p, miR-449a-5p, and miR-146a-5p were significantly up- and downregulated in the F compared to the S group, respectively. The SP from F and S rabbit males contains EVs with different miRNA cargo correlated with spermatogenesis, homeostasis, and infertility, which could be used as biomarkers for male fertility and potential therapies for assisted reproductive technologies.

Uterine issues in infertile queens: Nine cases.

This prospective case series investigated potential uterine causes of infertility in queens. Purebred queens with infertility (failure to conceive, embryonic death, or failure to maintain pregnancy and produce viable kittens), but no other reproductive disorders were examined approximately 1-8 weeks before mating (Visit 1), 21 days after mating (Visit 2), and 45 days after mating (Visit 3) if pregnant at Visit 2. Investigations included vaginal cytology and bacteriology, urine bacteriology, and ultrasonography. At Visit 2 or 3, uterine biopsy or ovariohysterectomy was performed for histology. Of nine eligible queens, seven were non-pregnant by ultrasound at Visit 2 and two had lost pregnancies by Visit 3. Ovulation was confirmed by serum progesterone concentration in all queens. Ultrasonic appearance of the ovaries and uterus was compatible with a healthy status except for one queen with signs of cystic endometrial hyperplasia (CEH) and pyometra, a follicular cyst in another, and fetal resorptions in two queens. Six cats had histologic lesions of endometrial hyperplasia, including CEH (n=1). Only one cat had no histologic uterine lesions. Bacteria were cultured from vaginal samples in seven queens at Visit 1, (two were non-evaluable), and in five of seven queens sampled at Visit 2. Uterine cultures were negative except for the cat with pyometra. All urine cultures were negative. In summary, the most frequent pathology observed in these infertile queens was histologic endometrial hyperplasia, which can potentially inhibit embryo implantation and healthy placental development. This suggests that uterine disease might contribute substantially to infertility in purebred queens.

Spermatogenesis regeneration by transfected spermatogonial stem cells in infertile roosters through testicular transplantation.

Investigations pertaining to spermatogonial stem cells (SSCs) have led to the use of these cells in a variety of fields including infertility treatments, production of transgenic animals, and genome editing. The aim of the present study was to investigate the plausibility of regenerating spermatogenesis in infertile roosters by transplanting transfected SSCs into testes. Spermatogonial stem cells were isolated and cultured for seven days. Afterward, pDB2, a plasmid vector carrying a reporter gene, GFP, was transfected into the SSCs. Transfected SSCs were transplanted into the left testis of infertile roosters. Tissue samples from the recipients' testes were obtained six weeks after the transplantation and transplanted SSCs were observed in the basement membrane. After eight weeks, GFP-positive spermatozoa were observed in collected semen from the recipient roosters and GFP gene in spermatozoa was confirmed using PCR. The recipient roosters were mated with hens. Hatchlings were visually checked and their tissue samples were tested by PCR to identify transgenesis but both of them were negative. Overall, it seems that regeneration of spermatogenesis in roosters via transfected SSCs is possible but more studies are need to produce recombinant proteins by this way.

Effect of different adjuvant formulations on the antibody response of horses to porcine zona pellucida proteins.

Immunization with porcine zona pellucida (PZP) proteins is being used successfully to induce infertility in wildlife including horses. However, widespread adoption of this method to control the growth of horse populations requires further refinement in order to induce long-term infertility, reduce the frequency and severity of injection site reactions, and make the vaccines easier to administer. The next generation of PZP-based vaccines will likely be a controlled-release formulation with different adjuvants from the Freund's adjuvants used in existing vaccines. We evaluated the response of equine peripheral blood mononuclear cells to a cationic nanoparticle adjuvant, Nano-11, alone and with the TLR agonists poly(I:C) and CpG ODN as a screen to develop an adjuvant system suitable for immunization of horses. The secretion of IL-1ß, TNF and CXCL10 were used as readouts. The combination of poly(I:C) with Nano-11 significantly increased the secretion of IL-1ß and TNF in comparison with Nano-11 only, with little effect of further addition of CpG ODN. The efficacy of the Nano-11/poly(I:C) adjuvant to enhance the immune response to native PZP proteins was determined in horses. Horses were immunized twice with the licensed Zonastat-H vaccine or PZP with Nano-11/poly(I:C) emulsified in silicone oil. A third group received PZP with the saponin adjuvant QA-21 emulsified in silicone oil. The horse sera collected monthly after the injections had increased anti-PZP IgG antibodies with the strongest response observed with Zonastat-H. We conclude that Nano-11/poly(I:C) is a potential candidate for the development of a controlled release formulation of a next generation PZP-based immunocontraception.

Comparison Between Gynecological Examination Methods and Sample Collection Techniques for the Diagnosis of Endometritis in Subfertile Mares.

Endometritis is a relevant cause of subfertility in mares. However, the accurate diagnosis, essential for effective treatment, can be difficult due to the variability of results and interpretations resulting from different examination methods and sample collection techniques. The present work compared gynecological evaluation methods and sample collection techniques to diagnose endometritis in subfertile mares. Forty animals with a history of subfertility were selected for gynecological evaluation using clinical methodologies, such as perineal conformation, transrectal palpation and ultrasonography, vaginoscopy, and digital examination of the cervix. In addition, we performed laboratory analyses, including uterine microbiological culture and endometrial cytology and histology, of which the latter is the gold standard for the diagnosis of endometritis. Samples were collected for microbiological culture and endometrial cytological evaluations using three different techniques: a commercial cytobrush/swab collector, low-volume uterine flush, and a new tested technique, by flush the fragment resulting from the endometrial biopsy. Transrectal palpation and ultrasound showed the best results among clinical examinations. However, they were less efficient in laboratory tests of endometrial cytology and uterine microbiological culture, in which the latter showed the highest sensitivity and specificity for endometritis compared with endometrial histology. The use of multiple results from different methods has also proved to be an effective alternative for diagnosis. Among the techniques used to collect endometrial material for cytology and microbiological culture, the most effective and practical in this study was the commercial cytobrush/swab collector.

Infertility In Queens: Clinical approach, experiences and challenges.

CLINICAL CHALLENGES: It is not easy for a veterinarian to determine the cause of infertility in a queen. The simplest method is to start by investigating the most common causes and then progress by successive elimination to the less common causes. For example, mating problems are commonly responsible for reproductive failure, and owners should be particularly vigilant in checking that the mating is progressing satisfactorily. Frequently, a veterinarian may have to determine, among other things, if there is a uterine or hormonal disorder or if an infectious pathology may be involved. In the case of a queen that is not cycling, it is important to differentiate a lack of onset of puberty in a young queen from a lack of cyclicity in an adult queen. AIM: This review sets out a practical, step-by-step approach that veterinarians can follow, starting with the most common causes of infertility in queens and moving towards more rare or specific causes. EQUIPMENT AND TECHNICAL SKILLS: Simple techniques such as vaginal smears are essential to determine if a queen that is ready to be mated is in full oestrus, if a queen is cycling or not and to diagnose inflammation of the genital tract. Veterinarians should be familiar with the peculiarities of the swabbing technique in cats, as well as the interpretation of feline vaginal cytology. Progesterone assays are useful on many occasions in the diagnosis pathway. A laboratory capable of performing karyotypes should ideally be available. Finally, it is important to be able to perform an ultrasound examination of the ovaries and uterus in a queen. EVIDENCE BASE: Although it appears that infertility is a problem among purebred queens, there is a lack of knowledge in this area and too few published studies. Some causes of feline infertility remain poorly investigated.

Exploiting 16S rRNA-based metagenomics to reveal neglected microorganisms associated with infertility in breeding bulls in Spanish extensive herds.

Bovine infectious infertility represents a problem due to the high impact on animal production and, in many cases, in public health. A lack of information on the characteristics of the bacterial population of the bovine reproductive system can hamper a comprehensive understanding of reproductive pathologies and the role that the microbiome could play. A metagenomic study based on the V3-V4 hypervariable region of the bacterial 16S rRNA gene was performed in 1029 preputial samples from bulls raised in an extensive regimen in Spain (944 from herds with low fertility rates -case group-, and 85 samples from reproductively healthy herds -control group-). The most representative phyla as well as the most 10 abundant bacterial families and their abundance did not show significant differences in both case and control groups. Similarly, the (alpha and beta) diversity of the bacterial populations was similar in both type of herds: the Shannon and Simpson indices show a high diversity of species, while the Bray-Curtis dissimilarity index did not show relevant differences in the bacterial communities. A deeper analysis of the operational taxonomic units showed the presence of one genera, Mycoplasma spp. significantly associated with fertility problems. Our study highlights the promising potential that the application of sequencing techniques (e.g. 16S rRNA-based metagenomics) possesses in examining bovine infertility, as they are able to reveal different pathogens that could go unnoticed using diagnostic approaches for only the main known pathogens.