Skip to main content

Advertisement

  • Research article
  • Open Access

Interaction of different Chlamydiae species with bovine spermatozoa

Contributed equally
BMC Microbiology201919:23

https://doi.org/10.1186/s12866-019-1392-z

  • Received: 17 January 2018
  • Accepted: 10 January 2019
  • Published:

Abstract

Background

Interaction of spermatozoa and Chlamydiae spp. might contribute to reduced fertility in cattle. To proof this hypothesis, bovine semen was incubated with viable or heat inactivated Chlamydia (C.) abortus or psittaci (Multiplicity of infection = 1) and sperm motility was monitored with a computer-assisted sperm analyzer over 24 h. Additionally, the interaction with the spermatozoa was further investigated by means of light and transmission electron microscopy (TEM).

Results

Only viable Chlamydiae of both species decreased sperm motility and this only after about 9 h. Taking binding rates into account, the loss of sperm motility after about 9 h could likely be a consequence of Chlamydiae attachment to the spermatozoa. About two thirds of the Chlamydiae elementary bodies were bound to the front third of the sperm, the acrosomal region. No inclusions of Chlamydiae in spermatozoa were observed in TEM after 2 h co-incubation.

Conclusions

As initial motility was not affected following co-incubation of viable Chlamydiae and bovine sperm, it seems likely that sperm could serve as a carrier/vehicle for Chlamydiae facilitating cervical passage of Chlamydiae spp. in cattle. Additionally, our results suggest that spermatozoa carrying Chlamydiae may have no initial disadvantage in reaching the oviduct, but are immotile at the time of ovulation what might have an impact on fertilization capacities of the individual sperm. Consequently, high concentrations of the investigated Chlamydiae in the seminal plasma or female genital tract might play a role in reduced fertility in cattle.

Keywords

  • Chlamydiae
  • Cattle
  • Semen motility
  • CASA (computer assisted sperm analysis)

Background

Chlamydia (C.) trachomatis infections are known to have a serious impact on the fertility of women, mainly by causing salpingitis [13]. In animals, Chlamydiae infections may remain asymptomatic, e.g. in cattle [4] or pigs [5], but they were also linked to several reproductive pathologies e. g. vaginitis and endometritis [57], mastitis and agalactia [5, 8], salpingitis [9], reproductive failure [5, 10] and abortion [11, 12].

Little is known about the role of Chlamydiae spp. in male. The pathogen was detected in semen of several species, like man, bull, ram and boar [1318]. Infections may cause urethritis and prostatitis [19, 20] but the influence of Chlamydiae on male fertility is still controversial. Co-incubation with C. trachomatis or chlamydial LPS was shown to cause sperm death [2123], likely due to increased apoptosis [23]. Some studies have demonstrated Chlamydiae infections to be correlated with reduced motility, reduced velocity and increased abnormal morphology of spermatozoa [2426]. In other studies, however, no significant impact on semen quality and fertility was found in man [27, 28], rat [29], boar [14, 18] and bull [13, 17].

Venereal infection is the classical route for the transmission of C. trachomatis in humans [3033]. There is limited evidence that venereal transmission of C. abortus is possible in sheep [15]. Recently, Schautteet et al. [16] reported severe reproductive failure in sows probably related to insemination of C. suis contaminated semen. Hamonic et al. [34] confirmed that C. suis remains viable and infectious during chilled storage and is more or less unaffected by antibiotics in extenders. The authors consequently hypothesized that extended semen may act as a viable transmission mechanism for C. suis in swine [33, 35]. Although it seems obvious that sperm can serve as a vector (vehicle) for Chlamydiae to infect the female genital tract, information on interactions between sperm cells and these bacteria is still patchy. There are two studies indicating that, after natural infection of man, C. trachomatis penetrates the sperm, preferentially their heads, and can also proliferate within the spermatozoa as indicated by the presence of reticulate bodies [33, 35]; however, the interaction for other host-Chlamydiae spp. has not been investigated yet.

To gain further insights into the role of Chlamydiae spp. in the bovine, we studied the interaction of C. abortus and psittaci, Chlamydiae spp. previously identified in semen samples of breeding bulls [13, 17], with bovine spermatozoa by means of light and transmission electron microscopy. Furthermore, we monitored the motility of spermatozoa in inoculated semen samples as a parameter directly correlated to fertility.

Methods

Animal and semen samples

Semen samples were obtained from an adult, sexually mature black Holstein Friesian bull housed in the Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with Veterinary Ambulance in Giessen, Germany (50°35’N 8°40’O). The bull was kept in a 22 m2 freestall barn under natural light conditions and temperatures ranging between 15 and 25 °C. He had ad libitum access to water, hay and straw and was additionally fed a commercial diet once a day according to the manufacturer’s instructions. Semen was collected using an artificial vagina (Minitube, Tiefenbach, Germany), and a cow or a bull as dummy. Immediately after collection, the semen samples were examined for sperm concentration as well as for total and progressive motility. For further experiments, samples were diluted to a concentration of 100 × 106 sperm/ml using 35 °C pre-warmed Biladyl® extender without antibiotics (Minitube).

Chlamydia stock solution

Chlamydiae were propagated on Buffalo-Green-Monkey-Kidney cells (ZBV Friedrich-Löffler-Institute, 17,493 Greifswald, Insel Riems, Germany), cultured in sterile filtrated medium [440 ml Eagle’s Minimum Essential Medium supplemented with 0.425 g NaHCO3, 5 ml 200 mM L-glutamine (Biochrom GmbH, Berlin, Germany), 5 ml Vitamin 100x (Biochrom GmbH) and 50 ml heat-inactivated fetal bovine serum (FBS, all Biochrom GmbH)] at 37 °C for about 4–7 days. After adding C. abortus or C. psittaci to the cells, vials were centrifuged for 1 h at 1935 rcf (centrifuge J2–21, rotor JS 7.5, Beckman Coulter GmbH, München, Germany) to promote infection of cells. Chlamydiae were allowed to grow for about 4–7 days at 37 °C before being harvested and separated from cell debris as described elsewhere [36]. Analysis with a transmission electron microscope (Zeiss EM 109, Oberkochen, Germany) revealed that the harvested pellet was mainly composed of elementary bodies. Chlamydiae concentrations were assessed by counting particles in Gimenez stained smears using an Ortholux II microscope with a counting tube (Leitz Wetzlar, Germany). Briefly, suspensions were diluted 1:50, 1:100 and 1:200 with sterile saline. 10 μl of each dilution were air-dried on a 1 cm2 area on a slide and fixated with 100% methanol (Sigma Aldrich, Seelze, Germany) for 1 h. Slides were incubated for 6 min in 0.5 ml carbol fuchsine solution (1.5 mg/ml Neofuchsine, Merck, Darmstadt, Germany, 3 mg/ml phenol, Merck, in phosphate buffer). Afterwards they were rinsed twice with water and counterstained with 0.5 ml malachite green solution (8 g malachite green, Merck, in 1000 ml distilled water) for a minute. Concentration of chlamydial particles was calculated based on the number of particles counted in 100 fields of a counting ocular considering optical magnification (787.5 fold).

Influence of Chlamydiae spp. on total and progressive motility

Ten [10] μl of either a suspension of viable Chlamydiae spp. (C. abortus or C. psittaci, 100 × 108 particles/ml; viability was confirmed by infection of Buffalo-Green-Monkey-Kidney cells), a corresponding suspension of heat-inactivated (95 °C, 1 h) Chlamydiae spp., or sterile saline were added to 100 × 106 spermatozoa diluted in 990 μl Biladyl®. The Chlamydia: sperm ratio corresponds to a multiplicity of infection (MOI) of 1. Samples were incubated at 35 °C for 24 h. Every three hours, 3 μl were then transferred into a 20-μl sample chamber (Leja® Standard Count 4 Chamber Slide, 20 μm, Leja Products B. V., Nieuw Vennep, Netherlands) for motility analysis at 37 °C. Percentages of motile and progressively motile spermatozoa were assessed as six repeated measurements with the computer-assisted sperm analyzer (CASA; SpermVisionTM Software Version 3.5.6.2; Minitube) using the settings as given in Table 1. All experiments were performed five times with semen from different preparations.
Table 1

Technical settings of the CASA system SpermVision™ for motility analysis

Parameter

Setting

Field-of-view depth = Depth of sample chamber

20 μm

Light adjustment

90–105

Total number of cells evaluated or number of fields

4000 spermatozoa or 8 fields

Sperm recognition area

22–99 μm2

Frame rate

60 frames/sec.

Points assessed for sperm motility

11

Total motility

progressive motility + local motility

Immotile sperm

AOC < 5°

Local motility

DSL < 6.0 μm

Progressive motility

Every cell that is not “immotile” or “local motile”

Hyperactive sperm

VCL > 80 μm/s, ALH > 6.5 μm and LIN < 0.65

Linear sperm

STR > 0.5 and LIN > 0.35

Non-linear sperm

STR ≤ 0.5 and LIN ≤ 0.35

Curvilinear sperm

DAP/Radius ≥ 3 and LIN < 0.5

Chlamydia-sperm interactions

To investigate the interactions of Chlamydiae spp. and bovine spermatozoa by light microscopy, 1 ml Biladyl® diluted spermatozoa- Chlamydiae suspension (100 × 106 sperm and respective number of Chlamydiae spp./ml, C. abortus or C. psittaci) were incubated at 35 °C for 24 h (long-term co-incubation). In 3-h intervals, 25 μl of the initial suspension were diluted with sterile saline up to 1 ml and centrifuged (tabletop centrifuge, 3 min, 4025 rcf). The supernatant containing unbound Chlamydiae was discarded. The pellet was re-suspended with sterile saline and centrifuged again. This centrifugation-washing procedure was performed five times in total. After the last centrifugation step (1935 rcf, 10 min, centrifuge JS-21, Rotor JS-7.5, Beckman Coulter GmbH), spermatozoa were transferred on a cover slide in a 6 ml polystyrene vial (Greiner Bio-One). Afterwards, slides were fixed with 1 ml 100% methanol (Sigma Aldrich) and Gimenez stained (see above). Using a Leica DMRIIC microscope (Leica, Wetzlar, Germany), 50 spermatozoa were examined at a magnification of × 1000. Presence (yes/no) and localization (acrosomal region/remaining other parts of the sperm) of Chlamydiae were recorded. The same experiment was performed with samples taken every 30 min over a total of 3 h (short-term co-incubation). All experiments were repeated four times.

For both Chlamydiae spp., the presence, localization, and size of chlamydial particles as well as signs of invasion into spermatozoa were further analyzed by electron microscopy. One ml of the above described Chlamydiae-sperm suspension (100 × 106 Biladyl® diluted spermatozoa and C. abortus or C. psittaci/ml) was incubated at 35 °C for 2 h. Removal of unbound Chlamydiae was performed as described above. For electron microscopic investigations, the resulting pellets (n = 5) were fixed for 12 h with 0.1 mol/l sodium cacodylate buffer (Merck) containing 6% glutaraldehyde (Plano, Wetzlar, Germany). Afterwards, the samples were centrifuged for 10 min at 447 rcf (tabletop centrifuge). The pellets were washed three times with 0.1 mol/l sodium cacodylate buffer. Briefly, they were fixed for 1 h with 1% osmium tetroxide (Plano), dehydrated with ethanol (Merck), embedded in epoxy resin (Plano), sectioned, stained with 0.2% lead citrate and 0.5% uranyl acetate using an Ultrastainer (Leica Reichert, Bensheim, Germany) and evaluated with a transmission electron microscope (Zeiss EM 109, Zeiss, Oberkochen, Germany) at 80 kV. Pure Chlamydia particles in Buffalo-Green-Monkey-Kidney cell culture treated in the same way as indicated above served as positive control.

Statistical analysis

For all parameters assessed, the different Chlamydiae spp., C. abortus and C. psittaci, were evaluated individually. In general, data was presented as mean ± standard deviation (SD). For statistical analysis, samples were evaluated as repeated measures over the different time points.

To test for a significant effect of Chlamydiae spp. on semen motility and progressive motility, respectively, a mixed-effect model for a four-factorial analysis of variance with repeated measures and equal cell sizes was used (fixed factors: treatment and time as repeated measures; random effects: ejaculate and replication; program BMDP8V). A two-way analysis of variance (factors: time and localization) with repeated measures (program BMDP2V) was performed to analyze binding sites of Chlamydiae on the sperm, i.e. at the acrosomal region of the sperm head versus any other region of the sperm cell, such as midpiece and tail. Calculations for short- and long-term co-incubation, 3 and 24 h, respectively, were performed separately, excluding the time point 0 (The time when the first sample was taken – time 0).

For all tests, the statistical software program package BMDP Release 8.1 was used [37]. Values were considered to be statistically significant at P ≤ 0.05.

Results

Influence of chlamydia on total and progressive motility

Sperm motility constantly decreased during the 24 h of observation in all experiments. The effect varied strongly between the different ejaculates. Saline controls showed a final reduction of total and progressive motility of 26–90% and 19–81% respectively (data not shown). Nevertheless, test sets from single ejaculates showed a high repeatability. The relative standard deviation within 36 duplicates (saline control, 4 different ejaculates, and 9 different time points) was 0.83%, only. For evaluation of the chlamydial influence on sperm motility, the results for total and progressive motility of the Chlamydiae-inoculated samples were related to the results of the respective saline controls (normalized) and presented as the % difference (Fig. 1). Heat inactivated Chlamydiae of both species did not significantly reduce sperm motility during the course of the experiment. Spermatozoa co-incubated with viable Chlamydiae spp. showed motility results comparable to saline controls in the first nine hours. Interestingly, at all later examination times the immotile fraction was increasingly higher than in the samples inoculated with heat inactivated Chlamydiae spp. This effect was statistically significant for both Chlamydiae spp. with P < 0.0001 after 24 h. The average reduction of the motile fraction was more distinct in samples inoculated with C. abortus (19%) than with C. psittaci (15%). This effect was even stronger in terms of the mean reduction of progressive motility (C. abortus 34%, and C. psittaci 18%).
Fig. 1
Fig. 1

Viable Chlamydia abortus und psittaci signicantly decrease a. total and b. progressive motility of bovine spermatozoa. Percentage of immotile bovine sperm (normalized against saline control) coincubated at 35 °C with viable and heat-inactivated Chlamydia (MOI = 1) for 24 h. Results are depicted as mean values ± SD of 5 replicates. Motility of spermatozoa co-incubated with viable Chlamydiae spp. was significantly reduced from 9 h onwards compared to motility of spermatozoa incubated with heat inactivated Chlamydiae (p < 0.0001)

Chlamydia sperm interaction

Light microscopy revealed that viable C. abortus and psittaci were both able to attach to bovine spermatozoa (Fig. 2) with no significant differences between the two investigated Chlamydiae spp.. Detailed results on the time course and localization of attachment of the two different Chlamydiae spp. studied are given in Fig. 3. Binding rates increased in a time-dependent manner. About two thirds of the Chlamydiae (in mean 60.3% of C. abortus and 62.9% of C. psittaci) were found to be attached to the acrosomal region of the sperm head comprising only one third of the sperm surface. In the course of the experiment, this ratio did not change significantly.
Fig. 2
Fig. 2

Light microscopical evaluation clearly confirms binding of C. abortus and psittaci to bovine spermatozoa. The orange particles represent chlamydial particles (a. C. abortus; b. C. psittaci) bound to a bovine spermatozoon. The particles look orange due to the use of a blue filter. In the negative control (c.), no similar particles are visible (magnification: × 1000)

Fig. 3
Fig. 3

C. abortus and psittaci can attach to bovine spermatozoa. The apical area of the sperm head seems to be the preferred location for attachment. Attachment of Chlamydia spp. (C. abortus, C. psittaci,) to the apical (acrosomal) area and other parts of bovine spermatozoa during 24 h of coincubation (MOI = 1) at 35 °C. Results are depicted as mean values of 4 replicates of 50 sperms ± SD. [The top error bar indicates the error bar of “the other parts” for the respective Chlamydia spp. (only positive SD presented); the lower error bar indicates the error bar of “the apical part” (only negative SD presented).] Approximately 60% of Chlamydiae spp. particles are attached to the apical area corresponding to the acrosomal region

Using transmission electron microscopy, Chlamydiae were visualized as small round particles of 0.3 to 0.4 μm attached to the spermatozoon’s surface (Fig. 4B). Double membranes surrounded the dark and electron dense particles allowing for easy differentiation from larger cytoplasmic droplets. Chlamydia particles in Buffalo-Green-Monkey-Kidney cell culture pellet were shown after harvesting as positive control (Fig. 4A). Similar to what has been observed with light microscopy (see Fig. 2), most of the Chlamydiae seemed to be located at the apical part of the sperm in the acrosomal area. No particles or structures strongly ressembling Chlamydiae could be identified inside the sperm head or its nucleus after 2 h of co-incubation by using TEM, nor were any changes in the acrosomal membrane of spermatozoon obvious at the attachment site following 2 h co-incubation.
Fig. 4
Fig. 4

Transmission electron microscopic images showing Chlamydia-sperm interaction

Exemplary chlamydial particles are marked. a. Chlamydial particles in Buffalo-Green-Monkey-Kidney cell culture after harvesting, size indicating elementary bodies, additionally cell debris visible and b. a Chlamydia spp. particle, resembling an elementary body localised at the apical part of the sperm head (all magnifications M12000, bar indicates 0.6 μm)

Discussion

The present data shows that viable Chlamydiae of both investigated species, C. abortus and C. psittaci, at an MOI of 1 are capable to affect motility of bovine sperm. The effect on motility, a reduction by 15–19%, as observed in this study, is rather impressive, as spermatozoa were inoculated with Chlamydiae spp. at an MOI of only 1 resulting in low attachment rates (Fig. 2). Data on natural chlamydial load in semen is rare and, to the best of our knowledge, is not available for bovine ejaculates. Low chlamydial loads, i.e. 1.5 × 104 [38] or 4.9 × 104 [39] particles, were detected in ejaculates of infected men by Real time-quantitative polymerase chain reaction (RT-qPCR). Due to the lack of details about sperm concentration in the aforementioned studies [3840], it is impossible to compare those with this study in terms of chlamydial load necessary to elicit effects on sperm. However, comparison with the WHO reported minimum and average sperm concentrations in the human ejaculate (9-73 × 106/mL; [41]) reveal possible MOIs between 2.1 × 10− 4 and 5.4 × 10− 3. Different to the likely low chlamydial load in semen, a higher chlamydial load might be expected in the female genital tract where up to 8.5 × 107 C. trachomatis per ml genital fluid had been found in women [39] indicating that similar chlamydial and sperm concentrations as used here, might be possible naturally.

Sperm motility started to decrease nine hours of co-incubation with the respective Chlamydiae spp.. In contrast binding of chlamydial particles to sperm was already microscopically observed immediately after the start of co-incubation, and was correlated with time. It seems possible that the negative effect of Chlamydiae spp. on total and progressive motility might be due to chlamydial viability, since this effect was absent when spermatozoa were co-incubated with saline only (negative control) or heat-inactivated Chlamydiae. Heat-inactivated Chlamydiae spp. showed a certain lightmicroscopical integrity and typical Gimenez-staining, however, detailed examinations on the morphology by transmission electron microscopy were not performed as well as details on binding partners/receptors were lacking. These analyses should be included in future studies to identify the binding mechanism. In contrast to this study, other investigations did not find such negative effects on semen parameters, which might be, at least in part, due to differences in the study designs. In particular, semen parameters were evaluated shortly after starting co-incubation (e.g. [27, 28, 35]) at a time where also in this study, no effects of Chlamydiae spp. on motility were observed.

Taken together, the results of the current study suggest that spermatozoa start to display a reduced motility around 9 h post co-incubation due to effects of Chlamydiae spp.. Moreover, C. abortus induced a slightly stronger effect than C. psittaci. The reasons for this difference remain to be investigated.

Basic knowledge of the sperms’ fate in the bovine female genital tract is necessary for understanding possible clinical effects of Chlamydiae-mediated reduced sperm motility on female reproduction. Following male infection, Chlamydiae spp. are located in the accessory sex glands in man [19, 20], bulls and boars [42]. During ejaculation, spermatozoa get in contact to the pathogen that is released from the accessory sex glands together with the seminal plasma. In bovines, the spermatozoa are deposited into the anterior vagina during breeding, and are then required to rapidly enter the cervix. However, the cervical passage of sperm is hampered by the cervical mucus, which acts as a mechanical barrier particularly to sperm with abnormal motility patterns or reduced motility thus being a mechanism of sperm selection [4346]. Additionally, the cervical mucus is also considered to have a filter function for seminal plasma and free microbes [47]. It might thus be that binding to motile spermatozoa is necessary for Chlamydiae spp. to not be caught by the cervical mucus in order to be able to reach the uterus and eventually also the oviduct. After having passed the cervix, spermatozoa quickly reach the utero-tubal junction where the apical surface of the sperm head binds to the oviductal epithelium [48, 49] at the site of sperm reservoir (for review see [47, 50]). At this site, sperm remain viable for 18 to 24 h or even longer as earlier studies have shown that artificial insemination with frozen semen (as common in cattle) is most successful, if it is performed in oestrus 12–24 h before ovulation [51]. As motility was first significantly affected after nine hours of co-incubation and the percentages of spermatozoa with bound spermatozoa are similar, it seems, however, likely that Chlamydiae–carrying spermatozoa are immotile at the time of fertilization. It remains to be investigated why motility was affected from nine hours after co-incubation and what were the reasons of immotility of spermatozoa. It is noteworthy that heat inactivated Chlamydiae spp. had no impact on sperm motility and it deserves further investigation if addition of specific antibiotics to commercial semen extenders or semen freezing is capable to induce the same effects on Chlamydiae sp.. in semen samples. The inactivation process might have resulted in denaturation of chlamydial structures relevant e. g. for the binding to the host cell, the observed reduction of motility occurred when Chlamydiae spp. in other host cells might start intracellular replication, energy parasitism and induction of other severe metabolic changes as a consequence of invasion [4, 52].

Our data did, however, not show an invasion of the studied Chlamydiae spp. into the bovine spermatozoa, only adhesion of 0.3–0.4 μm particles considered as infectious elementary bodies was visualised by means of light and transmission electron microscopy. Adherence of chlamydial particles to the sperm surface has been described before for C. trachomatis following in vitro [53] and in vivo infection [33, 35], with the latter authors also describing elementary and reticulate bodies within the sperm head [33, 35] and tail [33] by means of transmission and scanning electron microscopy. Interestingly, the described changes resemble previously described genetic or fixative-related membrane changes and defects, crater-like changes in the acrosome and chromatin defects in the sperm head (for review see [54]). It remains to be clarified if the lack of invasion into spermatozoa is real in this Chlamydiae spp. – sperm interaction or could have been related to the use of different Chlamydiae spp. (C. trachomatis versus C. abortus and C. psittaci) and hosts (human versus bovine), the duration of Chlamydiae-sperm interaction (2 h) or due to the fact that the experiments were based on in vitro co-incubation and not on natural infection.

Considering the attachment of Chlamydiae spp. to the sperm cells, seminal plasma proteins coating the spermatozoa deserve further attention as binding mediators. As about two thirds of the Chlamydiae particles were attached to the apical part of the sperm head, PDC-109 and osteopontin can be considered as strong candidates. Whereas PDC-109 modulates binding to the oviductal epithelium [5557] and is bound predominantly to the midpiece, but also to the acrosome as well as the post-equatorial and equatorial segments [57], osteopontin, predominantly identified on the acrosome, is hypothesized to be involved in sperm–oocyte interaction, thereby affecting fertilization [57].

Conclusions

Data shows that viable C. abortus and psittaci (MOI = 1) attach to spermatozoa. Initially, spermatozoa with attached Chlamydiae are not hampered. However, taking binding rates into account, our data also suggests that Chlamydiae spp. reduce sperm motility after 9 h of co-incubation in vitro, and thus possibly lead to a reduced fecundity of bull semen. As about two thirds of the Chlamydiae particles were bound to the front third of the sperm, the acrosomal region, it is suggested that specific binding proteins, like e.g. PDC-109, are involved in Chlamydiae-spermatozoa interaction.

Notes

Declarations

Acknowledgements

We gratefully thank the ZBV Friedrich-Löffler-Institute for provision of Chlamydiae to the Institute for Hygiene and Infectious Diseases of Animals and Franziska Sechser and Simone Herdt, Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals with Veterinary Ambulance in Giessen, Germany, for help with performing experiments and evaluation. The technical assistance from the technical staff from the Institute for Veterinary Anatomy, especially Susi Schubert-Porth, is gratefully acknowledged.

Funding

This study was founded by LOEWE-MIBIE (Initiative of the County Hesse for development of scientific-economic excellence, program: male infertility in case of infection and inflammation). The funding did not affect or influence the design of the study, the collection, analysis, and interpretation of data nor the writing of the manuscript.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authors’ contributions

AW, JK and SGP designed the study, TE did the experimental work, CH was responsible for the culture of Chlamydiae (partly together with TE) and critically discussed the results with SGP, MB did TEM analysis. KF performed all statistical analysis. SGP critically analyzed the data and wrote the initial draft of the manuscript. Manuscript revision was by AW, CH, MB and JK, finalized by SGP. All authors read and approved the final manuscript.

Ethics approval

This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The housing facilities and the protocol were approved by the Regierungspräsidium Giessen, Germany (AZ No. V 54-19c2015h02 GI 18/14 Nr. A 27/2012).

Consent for publication

Not applicable.

Competing interests

The authors declare no conflict of interest. I confirm that I have read BioMed Central’s guidance on competing interests and have included a statement indicating that none of the authors have any competing interests in the manuscript.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Klinikum Veterinärmedizin, Clinic for Obstetrics, Gynecology and Andrology of Large and Small Animals, Justus-Liebig University Giessen, Frankfurter Strasse 106, 35392 Giessen, Germany
(2)
Institute for Veterinary-Physiology and Biochemistry, Justus-Liebig University Giessen, Frankfurter Strasse 100, 35392 Giessen, Germany
(3)
Department of Veterinary Clinical Sciences, Section for Veterinary Reproduction and Obstetrics, University of Copenhagen, Dyrlægevej 68, 1870 Frederiksberg C, Denmark
(4)
Reproductive Unit of the Clinics – Clinic for Small Animals, University of Veterinary Medicine Hannover, Bünteweg 15, 30559 Hannover, Germany
(5)
Institute for Hygiene and Infectious Diseases of Animals, Justus-Liebig University Giessen, Frankfurter Strasse 85-89, 35392 Giessen, Germany
(6)
Institute for Veterinary Anatomy, Histology and Embryology, Justus-Liebig University Giessen, Frankfurter Strasse 98, 35392 Giessen, Germany
(7)
Ambulatorische und Geburtshilfliche Tierklinik, Veterinärmedizinische Fakultät, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany
(8)
Unit for Biomathematics and Data Processing, Justus-Liebig University Giessen, Frankfurter Str., 35392 Giessen, Germany

References

  1. Eggert-Kruse W, Gerhard I, Naher H, Tilgen W, Runnebaum B. Chlamydial infection-a female and/or male infertility factor? Fertil Steril. 1990;53:1037–43.View ArticleGoogle Scholar
  2. Wolner-Hanssen P, Mardh PA, Moller B, Westrom L. Endometrial infection in women with chlamydial salpingitis. Sex Transm Dis. 1982;9:84–8.View ArticleGoogle Scholar
  3. Wolner-Hanssen P, Westrom L, Mardh PA. Perihepatitis and chlamydial salpingitis. Lancet. 1980;1:901–3.View ArticleGoogle Scholar
  4. Reinhold P, Sachse K, Kaltenboeck B. Chlamydiaceae in cattle: commensals trigger organisms or pathogens? Vet J. 2011;189:257–67.View ArticleGoogle Scholar
  5. Schautteet K, Vanrompay D. Chlamydiaceae infections in pig. Vet Res. 2011;42:29.View ArticleGoogle Scholar
  6. Blumer S, Greub G, Waldvogel A, Hassig M, Thoma R, Tschuor A, Pospischil A, Borel N. Waddlia Parachlamydia and Chlamydiaceae in bovine abortion. Vet Microbiol. 2011;152:385–93.View ArticleGoogle Scholar
  7. Wittenbrink MM. Detection of antibodies against chlamydia in swine by an immunofluorescent test and an enzyme immunoassay. Berl Münch Tierärztl Wochenschr. 1991;104:270–5.PubMedGoogle Scholar
  8. Kaltenboeck B, Kousoulas KG, Storz J. Detection and strain differentiation of chlamydia psittaci mediated by a two-step polymerase chain reaction. J Clin Microbiol. 1991;29:1969–75.PubMedPubMed CentralGoogle Scholar
  9. Longbottom D. Chlamydial infections of domestic ruminants and swine: new nomenclature and new knowledge. Vet J. 2004;168:9–11.View ArticleGoogle Scholar
  10. Wittenbrink MM, Kirpal G, Thiele D, Fischer D, Krauss H, Bisping W. Detection of chlamydia psittaci in vaginal discharge of cows: a necessary enlargement of bacteriologic diagnosis for the etiologic clarification of fertility disorders in the female cow. Zentralbl vetmed B. 1994;41:492–503.Google Scholar
  11. Griffiths PC, Plater JM, Martin TC, Hughes SL, Hughes KJ, Hewinson RG, Dawson M. Epizootic bovine abortion in a dairy herd: characterization of a chlamydia psittaci isolate and antibody response. Brit Vet J. 1995;151:683–93.View ArticleGoogle Scholar
  12. Pospischil A, Thoma R, von Bomhard W, Reitt K, Cantieni J, Zimmermann D, Polkinghorne A. Abortion in cattle caused by chlamydia psittaci. Schweizer Arch Tierheilkd. 2002;144:467–72.View ArticleGoogle Scholar
  13. Kauffold J, Henning K, Bachmann R, Hotzel H, Melzer F. The prevalence of chlamydiae of bulls from six bull studs in Germany. Anim Reprod Sci. 2007;102:111–21.View ArticleGoogle Scholar
  14. Kauffold J, Melzer F, Henning K, Schulze K, Leiding C, Sachse K. Prevalence of chlamydiae in boars and semen used for artificial insemination. Theriogenology. 2006;65:1750–8.View ArticleGoogle Scholar
  15. Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic implications. J Comp Pathol. 2003;128:217–44.View ArticleGoogle Scholar
  16. Schautteet K, De Clercq E, Miry C, Van Groenweghe F, Delava P, Kalmar I, Vanrompay D. Tetracycline-resistant chlamydia suis in cases of reproductive failure on Belgian Cypriote and Israeli pig production farms. J Med Microbiol. 2013;62:331–4.View ArticleGoogle Scholar
  17. Teankum K, Pospischil A, Janett F, Brugnera E, Hoelzle LE, Hoelzle K, Weilenmann R, Zimmermann DR, Gerber A, Polkinghorne A, Borel N. Prevalence of chlamydiae in semen and genital tracts of bulls rams and bucks. Theriogenology. 2007;67:303–10.View ArticleGoogle Scholar
  18. Teankum K, Pospischil A, Janett F, Burgi E, Brugnera E, Hoelzle K, Polkinghorne A, Weilenmann R, Zimmermann DR, Borel N. Detection of chlamydiae in boar semen and genital tracts. Vet Microbiol. 2006;116:149–57.View ArticleGoogle Scholar
  19. Wagenlehner FM, Weidner W, Naber KG. Chlamydial infections in urology. World J Urol. 2006;24:4–12.View ArticleGoogle Scholar
  20. Weidner W, Wagenlehner FM, Diemer T. Chlamydia infections of the male: what is relevant for urologists? Urologe A. 2006;45:1504–8.View ArticleGoogle Scholar
  21. Eley A, Hosseinzadeh S, Hakimi H, Geary I, Pacey AA. Apoptosis of ejaculated human sperm is induced by co-incubation with chlamydia trachomatis lipopolysaccharide. Hum Reprod. 2005;20:2601–7.View ArticleGoogle Scholar
  22. Hosseinzadeh S, Pacey AA, Eley A. Chlamydia trachomatis-induced death of human spermatozoa is caused primarily by lipopolysaccharide. J Medic Microbiol. 2005;52:193–200.View ArticleGoogle Scholar
  23. Satta A, Stivala A, Garozzo A, Morello A, Perdichizzi A, Vicari E, Salmeri M, Calogero AE. Experimental chlamydia trachomatis infection causes apoptosis in human sperm. Hum Reprod. 2006;21:134–7.View ArticleGoogle Scholar
  24. Al-Sweih NA, Al-Fadli AH, Omu AE, Rotimi VO. Prevalence of chlamydia trachomatis mycoplasma hominis mycoplasma genitalium and Ureaplasma urealyticum infections and seminal quality in infertile and fertile men in Kuwait. J Androl. 2012;33:1323–9.View ArticleGoogle Scholar
  25. Sellami H, Znazen A, Sellami A, Mnif H, Louati N, Zarrouk SB, Keskes L, Rebai T, Gdoura R, Hammami A. Molecular detection of chlamydia trachomatis and other sexually transmitted bacteria in semen of male partners of infertile couples in Tunisia: the effect on semen parameters and spermatozoa apoptosis markers. PLoS One. 2014;9:e98903.View ArticleGoogle Scholar
  26. Veznik Z, Pospisil L, Svecova D, Zajicova A, Unzeitig V. Chlamydiae in the ejaculate: their influence on the quality and morphology of sperm. Acta Obstet Gynecol Scand. 2004;83:656–60.View ArticleGoogle Scholar
  27. Filipiak E, Marchlewska K, Oszukowska E, Walczak-Jedrzejowska R, Swierczynska-Cieplucha A, Kula K, Slowikowska-Hilczer J. Presence of aerobic micro-organisms and their influence on basic semen parameters in infertile men. Andrologia. 2015;47:826–31.View ArticleGoogle Scholar
  28. Liu J, Wang Q, Ji X, Guo S, Dai Y, Zhang Z, Jia L, Shi Y, Tai S, Lee Y. Prevalence of Ureaplasma Urealyticum mycoplasma Hominis chlamydia trachomatis infections and semen quality in infertile and fertile men in China. Urology. 2014;83:795–9. https://doi.org/10.1016/j.urology.2013.11.009.View ArticlePubMedGoogle Scholar
  29. Motrich RD, Sanchez L, Maccioni M, Mackern-Oberti JP, Rivero VE. Male rat genital tract infection with chlamydia muridarum has no significant consequence on male fertility. J Urol. 2012;187:1911–7.View ArticleGoogle Scholar
  30. Debattista J, Timms P, Allan J, Allan J. Immunopathogenesis of chlamydia trachomatis infections in women. Fertil Steril. 2003;79:1273–87.View ArticleGoogle Scholar
  31. Gonzales GF, Munoz G, Sanchez R, Henkel R, Gallegos-Avila G, Diaz-Gutierrez O, Vigil P, Vasquez F, Kortebani G, Mazzolli A, Bustos-Obregon E. Update on the impact of chlamydia trachomatis infection on male fertility. Andrologia. 2004;36:1–23.View ArticleGoogle Scholar
  32. Paavonen J. Chlamydia trachomatis infections of the female genital tract: state of the art. Ann Med. 2012;44:18–28.View ArticleGoogle Scholar
  33. Erbengi T. Ultrastructural observations on the entry of chlamydia trachomatis into human spermatozoa. Hum Reprod. 1993;8:416–21.View ArticleGoogle Scholar
  34. Hamonic G, Pasternak JA, Käser T, Meurens F, Wilson HL. Extended semen for artificial insemination in swine as a potential transmission mechanism for infectious chlamydia suis. Theriogenology. 2016;86:949–56.View ArticleGoogle Scholar
  35. Vigil P, Mo Hamonic G, Pasternak JA, Käser T, Meurens F, Wilson HL. Extended semen for artificial insemination in swine as a potential transmission mechanism for infectious chlamydia suis. Theriogenology. 2016;86:949–56.View ArticleGoogle Scholar
  36. Pantchev A, Sting R, Bauerfeind R, Tyczka J, Sachse K. Detection of all Chlamydophila and chlamydia spp of veterinary interest using species-specific real-time PCR assays. Comp Immunol Microbiol Infect Dis. 2010;33:473–84.View ArticleGoogle Scholar
  37. Dixon WJ. BMDP statistical software manual volume 1 and 2, University of California press Berkeley Los Angeles London; 1993.Google Scholar
  38. Al-Mously N, Cross NA, Eley A, Pacey AA. Real-time polymerase chain reaction shows that density centrifugation does not always remove chlamydia trachomatis from human semen. Fertil Steril. 2009;92:1606–15.View ArticleGoogle Scholar
  39. Dirks JA, Wolffs PF, Dukers-Muijrers NH, Brink AA, Speksnijder AG, Hoebe CJ. Chlamydia trachomatis load in population-based screening and STI-clinics: implications for screening policy. PLoS One. 2015;10:e0121433.View ArticleGoogle Scholar
  40. Al-Moushaly A. Considerations on male infertility in genital infections with chlamydia trachomatis (CT). J Med Life. 2013;6:283–6.PubMedPubMed CentralGoogle Scholar
  41. Cooper TG, Noonan E, von Eckardstein S, Auger J, Baker HWB, Behre HM, Haugen TB, Kruger T, Wang C, Mbizvo MT, Vogelsong KM. World Health Organization reference values for human semen characteristics. Hum Reprod Update. 2010;16:231–45.View ArticleGoogle Scholar
  42. Storz J, Carroll EJ, Stephenson EH, Ball L, Eugster AK. Urogenital infection and seminal excretion after inoculation of bulls and rams with chlamydiae. Am J Vet Res. 1976;37:517–20.PubMedGoogle Scholar
  43. Barros C, Vigil P, Herrera E, Arguello B, Walker R. Selection of morphologically abnormal sperm by human cervical mucus. Arch Androl. 1984;12(Suppl):95–107.PubMedGoogle Scholar
  44. Hanson FW, Overstreet JW. The interaction of human spermatozoa with cervical mucus in vivo. Am J Obstet Gynecol. 1981;140:173–8.View ArticleGoogle Scholar
  45. Katz DF, Morales P, Samuels SJ, Overstreet JW. Mechanisms of filtration of morphologically abnormal human sperm by cervical mucus. Fertil Steril. 1990;54:513–6.View ArticleGoogle Scholar
  46. Katz DF, Slade DA, Nakajima ST. Analysis of pre-ovulatory changes in cervical mucus hydration and sperm penetrability. Adv Contracept. 1997;13:143–51.View ArticleGoogle Scholar
  47. Suarez SS. Interactions of spermatozoa with the female reproductive tract: inspiration for assisted reproduction. Reprod Fertil Develop. 2007;19:103–10.View ArticleGoogle Scholar
  48. Ignotz GG, Cho MY, Suarez SS. Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir. Biol Reprod. 2007;77:906–13.View ArticleGoogle Scholar
  49. Ignotz GG, Lo MC, Perez CL, Gwathmey TM, Suarez SS. Characterization of a fucose-binding protein from bull sperm and seminal plasma that may be responsible for formation of the oviductal sperm reservoir. Biol Reprod. 2001;64:1806–11.View ArticleGoogle Scholar
  50. Lefebvre R, Chenoweth PJ, Drost M, LeClear CT, MacCubbin M, Dutton JT, Suarez SS. Characterization of the oviductal sperm reservoir in cattle. Biol Reprod. 1995;53:1066–74.View ArticleGoogle Scholar
  51. Roelofs JB, Graat EA, Mullaart E, Soede NM, Voskamp-Harkema W, Kemp B. Effects of insemination-ovulation interval on fertilization rates and embryo characteristics in dairy cattle. Theriogenology. 2006;66:2173–81.View ArticleGoogle Scholar
  52. Hatch TP, Al-Hossainy E, Silverman JA. Adenine nucleotide and lysine transport in chlamydia psittaci. J Bactertiol. 1982;150:662–70.Google Scholar
  53. Wolner-Hanssen P, Mardh PA. In vitro tests of the adherence of chlamydia trachomatis to human spermatozoa. Fertil Steril. 1984;42:102–7.View ArticleGoogle Scholar
  54. Pesch S, Bergmann M. Structure of mammalian spermatozoa in respect to viability fertility and cryopreservation. Micron. 2006;37:597–612.View ArticleGoogle Scholar
  55. Gwathmey TM, Ignotz GG, Mueller JL, Manjunath P, Suarez SS. Bovine seminal plasma proteins PDC-109 BSP-A3 and BSP-30-kDa share functional roles in storing sperm in the oviduct. Biol Reprod. 2006;75:501–7.View ArticleGoogle Scholar
  56. Gwathmey TM, Ignotz GG, Suarez SS. PDC-109 (BSP-A1/A2) promotes bull sperm binding to oviductal epithelium in vitro and may be involved in forming the oviductal sperm reservoir. Biol Reprod. 2003;69:809–15.View ArticleGoogle Scholar
  57. Souza CE, Moura AA, Monaco E, Killian GJ. Binding patterns of bovine seminal plasma proteins A1/A2 30 kDa and osteopontin on ejaculated sperm before and after incubation with isthmic and ampullary oviductal fluid. Anim Reprod Sci. 2008;105:72–89.View ArticleGoogle Scholar

Copyright

© The Author(s). 2019

Advertisement