Next Article in Journal
Enhancing Smart Home Security: Anomaly Detection and Face Recognition in Smart Home IoT Devices Using Logit-Boosted CNN Models
Next Article in Special Issue
Using the Photo–Piezoelectric Effect of AuPt@BaTiO3 Oxidase Mimetics for Colorimetric Detection of GSH in Serum
Previous Article in Journal
Ecological Fall Prediction Sensitivity, Specificity, and Accuracy in Patients with Mild Cognitive Impairment at a High Risk of Falls
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Sensors
Volume 23
Issue 15
10.3390/s23156978
sensors-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Strategies for Bacterial Eradication from Human and Animal Semen Samples: Current Options and Future Alternatives

by
Michal Ďuračka
1,
Filip Benko
2,3,
Milan Chňapek
3 and
Eva Tvrdá
3,*
1
AgroBioTech Research Centre, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia
2
Institute of Applied Biology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia
3
Institute of Biotechnology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia
*
Author to whom correspondence should be addressed.
Sensors 2023, 23(15), 6978; https://doi.org/10.3390/s23156978
Submission received: 30 June 2023 / Revised: 2 August 2023 / Accepted: 4 August 2023 / Published: 6 August 2023
(This article belongs to the Special Issue Editorial Board Members' Collection Series: Biosensors Based on Nanotechnology: Fabrication, Structure and Application)
Browse Figures
Versions Notes

Abstract

:
The primary role of semen processing and preservation is to maintain a high proportion of structurally and functionally competent and mature spermatozoa, that may be used for the purposes of artificial reproduction when needed, whilst minimizing any potential causes of sperm deterioration during ex vivo semen handling. Out of a multitude of variables determining the success of sperm preservation, bacterial contamination has been acknowledged with an increased interest because of its often unpredictable and complex effects on semen quality. Whilst antibiotics are usually the most straight-forward option to prevent the bacterial contamination of semen, antimicrobial resistance has become a serious threat requiring widespread attention. As such, besides discussing the consequences of bacteriospermia on the sperm vitality and the risks of antibiotic overuse in andrology, this paper summarizes the currently available evidence on alternative strategies to prevent bacterial contamination of semen prior to, during, and following sperm processing, selection, and preservation. Alternative antibacterial supplements are reviewed, and emphasis is given to modern methods of sperm selection that may be combined by the physical removal of bacteria prior to sperm preservation or by use in assisted reproductive technologies.

1. Introduction

Early attempts for sperm vitality conservation may be traced back to 1776, when Spallanzani froze spermatozoa using snow and subsequently demonstrated their motility after rewarming [1]. Since then, remarkable progress in the evolution of techniques and media for short- or long-term semen storage has allowed sperm preservation to overcome numerous space and time limitations, and thus to become an integral part of assisted reproduction technologies (ARTs) in animals and humans [1,2].
Sperm preservation, at low temperatures and when properly performed, enables the long-term storage of male gametes in a state of metabolic arrest that prevents cellular senescence while maintaining their viability and fertilizing potential, therefore this allows them to be used when and where they are needed [2]. While the role of sperm storage at low temperatures as a critical pillar of reproductive technologies is undoubtable, the rationale behind its use for the management of male fertility may differ between animal and human ARTs [3,4,5,6,7]. In humans, sperm storage represents an effective strategy to preserve male reproductive capacity following cytotoxic or surgical treatment that may cause possible testicular or ejaculatory dysfunction. Cryopreservation may also be routinely used in men who are exposed to potentially toxic agents which may interfere with spermatogenesis, or who begin assisted reproduction treatment, and thus have a back-up sperm source [3,4]. In donor insemination programs, sperm preservation is necessary to have enough time to screen the donors for possible exclusion criteria, before the semen sample is used for clinical purposes [4]. In animals, sperm preservation techniques are extensively employed to manage or accelerate the rate of genetic improvement, by using a lower number of studs with valuable traits to inseminate a large number of females, leading to a significant reduction in the cost of the breeding process [5,6]. Semen storage is also a valuable tool for the protection of genetic resources and biodiversity of protected or endangered animal species [7].
Although an important benefit of artificial insemination (AI) lies in the prevention of disease transmission, potential pathogens may be easily disseminated through an often-overlooked contaminated semen [8]. The term “bacteriospermia” refers to the presence of bacteria in the ejaculate [9] and is regarded as clinically relevant when the bacterial count exceeds 1000 colony-forming units (CFU)/mL semen [10]. In most cases, the condition is often associated with an acute or chronic bacterial infection of one or more organs of the male urogenital system, including the testes, epididymides, prostate, ureter, or urethra [8]. Gram-positive, Gram-negative, as well as Gram-unstainable bacteria may be regarded as causative agents for bacteriospermia associated with urogenital infection, out of which Escherichia coli, Staphylococcus aureus, Ureaplasma urealyticum, Chlamydia trachomatis, Mycoplasma genitalium, Enterococcus faecalis, and streptococci have received the most attention [10,11]. At the same time, large quantities of bacteria may be present even in semen samples collected from otherwise healthy and fertile subjects [12,13,14,15,16,17], since the male urogenital system naturally houses an inherent bacteriome [18,19]. Semen may also become contaminated by bacteria present in the prepuce and penile foreskin during ejaculation [20]. The bacterial contamination of particularly animal semen may also stem from skin, wool, bedding, or excretions [21]. Moreover, semen collection and processing procedures may not be per se carried out in an antiseptic environment, thus ejaculates may be infested by bacteria proceeding from electroejaculators or artificial vaginas, laboratory glassware, contaminated semen extenders and ART media, or poor on-site hygiene standards [21,22].
Under ideal circumstances, semen extenders provide protection to male gametes and preserve their motion and fertilizing ability over time, primarily by stabilizing the plasma membrane, providing sources of energy, and preventing any harmful effects of pH and osmolarity fluctuations or oxidative stress on spermatozoa [23]. On the other hand, sperm preservation media may act as a rich reservoir of nutrients and a favorable environment for bacterial growth and multiplication if a contaminated ejaculate is processed for ART or AI [24]. Although bacterial growth largely depends on the temperature, some bacteria are capable of growing, to some extent, even during cooling and/or freezing [25]. What is more, bacteria may survive even in frozen semen samples, resuming growth shortly following thawing [24]. In this sense, the time necessary for semen processing, dilution, cooling, freezing, and thawing should be kept to a minimum and strict hygiene measures should always be followed.
While the possibility of the initial bacterial contamination of semen is currently not taken into consideration by the current guidelines, neither for human nor for animal ejaculates [26], a vast collection of evidence indicates that bacteriospermia may lead to depleted sperm motion activity and membrane integrity [12,13,14,15,16,17,27,28], morphological aberrations to the sperm head, acrosome, mid-piece, and tail [10,13,14,27,28,29,30], a deteriorated mitochondrial function and ATP production [13,14,15,16,17,30], DNA fragmentation, and the onset of cell death [13,14,15,16,17,27,28,29,30]. Sperm agglutination [28,30,31], the release of bacterial toxins and cytotoxic cytokines, as well as high levels of reactive oxygen species (ROS) have also been frequently reported in ejaculates compromising bacteria [13,14,15,16,17,27,31]. It has also been revealed that the processing of a contaminated semen sample could modify the biochemical properties of semen extenders or ART media, which may compromise the shelf life of extended semen [22,26,32]. Subsequently, the use of bacteriospermic samples for AI may lead to the onset of various infections in the female [33,34,35] and contribute to lower embryo survival and pregnancy outcomes [36,37].
Based on the severity of consequences bacteriospermia may cause in the field of andrology and reproductive biology, this review discusses the strategies employed to prevent or manage the spread and persistence of bacteria in ejaculates, starting from conventional and currently employed approaches to alternative options with a promising perspective for the future.

2. Antibiotics

Antibiotics are currently the most preferred option for bacterial control during semen processing and storage, primarily because of their affordability, diversity, biological activity, and availability [38].
In the case of bovine and porcine ejaculates, Directives 88/407 and 90/429 of the European Council, Annex C2 state that an effective combination of antimicrobials, especially against mycoplasmas and leptospires, should be supplemented to each extended semen sample [39,40]. This combination must lead to an effect equivalent to 500 µg streptomycin/mL final dilution; 500 IU penicillin/mL final dilution; 150 µg lincomycin/mL final dilution; and/or 300 µg spectinomycin/mL final dilution. This primary legislative document was later supported by the Directive 92/65 [41], which is relevant to other animal species such as small ruminants, poultry, and Equidae, and which reports another combination of antimicrobials consisting of 50 μg tylosin/mL, 250 μg gentamicin/mL, and 150/300 μg lincomycin/spectinomycin/mL; or 25 μg divecacin/mL and 75 μg amikacin/mL, which may be used alone or in combination [41]. A similar approach is usually followed in human andrology, although no specific guidelines have been published by the World Health Organization (WHO) laboratory manual for the examination and processing of human semen [42].
The antibiotic substances that are most frequently used in semen diluents include β-lactams (penicillins, cephalosporins), whose mechanism of action lies in the inhibition of the bacterial cell wall synthesis, leading to cell lysis and death. Others include inhibitors of bacterial protein synthesis such as aminoglycosides (streptomycin, gentamicin, kanamycin), lincosamides (lincomycin) or macrolides (tylosin, tilmicosin, spectinomycin). Nevertheless, a large variation exists among different species with respect to the antimicrobial types and doses added to extenders used for both semen chilling and freezing procedures [43]. Furthermore, the exact combination and/or concentration of antibiotics in commercially available semen diluents and ART media is generally undisclosed by the producer.
Nevertheless, a convincing body of evidence gathered from recent studies emphasizes the presence of diverse bacterial strains found in native semen, insemination doses, ART media with concerning resistance patterns to antibiotics routinely used in andrology, animal production, and reproductive medicine (Table 1). These recurrent patterns may be accompanied by the horizontal or vertical transmission of bacterial resistance, which may pose a concerning threat to public health, the food chain, and the stability of ecosystems [44]. Furthermore, several studies have reported the significant negative effect of antibiotic supplements, such as gentamicin, streptomycin-penicillin, ciprofloxacin or doxycycline, on sperm motility, membrane, and DNA integrity as well as overall fertilization ability, stressing the need for the prudent use of antibiotics during sperm preservation [45,46,47]. Thus, the search for alternative options that would eliminate bacteria from semen by different means, while preventing the spread of bacterial resistance and additional cytotoxicity, has become a reality. Specific approaches will be discussed in detail in the following sections.

3. Antimicrobial Proteins, Biocompounds, Plant Extracts, and Nanoparticles

More than 5500 antimicrobial peptides (AMPs) are known for their effects against pathogens. Despite their wide structural diversity, most AMPs contain polar and non-polar parts in their structure [61]. Although the mechanism of action of AMPs is only partially investigated, most of these have studied bacteriostatic peptides belonging to the class of cationic amphipathic helices [62]. The advantage of this class is that these peptides show favorable selectivity against microbes when compared to hydrophobic and anionic peptides. Magainin is the most studied cationic peptide, consisting of 23 amino acids, and is currently used as a therapeutic [63]. Only a few studies reported the use of AMPs in semen storage. Hensel et al. [64] tested the effect of seven short cationic lipopeptides on porcine spermatozoa. Two lipopeptides did not show significant impairment of sperm quality while being antibacterial against several bacteria including Alcaligenes faecalis, Escherichia coli, Pseudomonas aeruginosa, Treuperella pyogenes, Pasteurella sp., Providencia stuartii, and Streptococcus porcinus. The membrane selectivity of AMPs lies in two specific factors: the first—an electrostatic interaction between the cationic peptide and high-acid bacterial membrane, and the second—high cholesterol amount in eucaryotic membranes stabilizes and makes them more rigid when compared to procaryotic membranes [65]. According to Bussalleu et al. [66], PMAP-37 at 3 µM did not affect boar spermatozoa quality while being effective against bacteria. The advantages of AMPs lie in their thermostability, economic availability, and ability to minimize the risk of antimicrobial resistance emergence [67]. On the other hand, there are several reasons why the use of naturally obtained AMPs is limited: susceptibility to proteases, cytotoxicity to host cells, inactivity at physiological salt concentrations, and sustainability of production. Therefore, the focus has shifted to the development of synthetic AMPs [68]. As AMPs and conventional antibiotics synergize antimicrobial activity, future strategies should deal with their mutual use. The combination of AMPs and antibiotic supplementation could reduce the minimal inhibitory concentration and cell toxicity.
Numerous studies have already pointed out that bioactive compounds may improve the oxidative milieu of stored semen, thus prolonging sperm survival in human [69], boar [70], bull [71], ram [72], and rooster semen [73]. In particular, flavonoids are the most important class of phenolic compounds exerting extraordinary antibacterial activity against multi-drug resistant bacterial strains [74]. Several mechanisms are behind their antibacterial effect, including the suppression of nucleic acid synthesis, membrane function, and energetic metabolism [75]. Moreover, bacterial adhesion, the formation of porins, and biofilm formation were reduced in the presence of flavonoids [76]. As previously noticed, the use of bioactive substances as antimicrobial supplements in semen storage is limited by their cytotoxic effects at high concentrations. The combination of carvacrol and thymol was found to be effective against present bacterial strains in stored boar semen (17 °C) without any significant effect on sperm motility and viability [77]. Amongst the flavonoids, 15 mg/L of icariin showed both antioxidant and antimicrobial effects in stored (17 °C) boar semen, while a combination with 62.5 mg/L of gentamicin instead of 250 mg/L showed similar effects on bacterial inhibition [78]. In the meantime, curcumin at a concentration of 5% significantly reduced the number of bacterial colonies within 2 h in stored bull semen and did not negatively influence spermatozoa.
Moreover, plant extracts also showed favorable effects on stored semen [79]; similarly to pure bioactive substances, their antibacterial effect in the storage medium could be as effective as sperm resistance to their cytotoxic effect at high concentrations. Schizandra chinensis extract presented with a minimal inhibitory concentration at 64.2 µg/mL which was effective against Enterococcus faecalis and Listeria monocytogenes and still did not negatively affect the functional activity and structural integrity of stored bull spermatozoa. However, most of the studied bacteria were susceptible to concentrations higher than 75 µg/mL, which could act as a prooxidant. Rosemary was previously reported to have an antibacterial effect in the form of essential oil in stored boar semen [80]. On the other hand, rosmarinic acid did not reveal any antibacterial activity against bacteria in bull semen, which indicates that the antibacterial activity of supplements stemming from plant sources may vary depending on their form.
In recent years, nanoparticles were found to be interesting antibacterial agents. However, only a few studies have been published concerning semen storage. A slight antibiotic effect with no detrimental effects on the sperm quality was observed when iron oxide nanoparticles were added [81]. The desired antibacterial effect was recorded when iron and silver nanoparticles were combined at the cost of increased spermatotoxicity [82]. Yousef et al. [83] disclosed that concentrations ≤30 µg/mL of silver-carbon nanoparticles did not affect sperm quality parameters during bull semen storage and were effective against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, although transmission electron microscopy revealed that these nanoparticles were attached to the plasma membrane but did not penetrate the bull sperm cell. However, it is not known how the nanoparticles would affect the recipients’ genitalia and embryo production. Finally, chitosan-nanoparticles, combined with ethylenediaminetetraacetic acid (EDTA) and bestatin, provided a bacteriostatic effect against Enterobacteriaceae without damaging rabbit spermatozoa stored at 15 °C for 72 h [84].

4. Centrifugation Techniques

Repeated washing and centrifuging of a large volume of semen in a culture medium represents the simplest removal technique of bacteria present in the seminal fluid. Usually, 2–3 centrifugations (not greater than 800× g) after mixing with 5–10× of culture medium are recommended [85]. The disadvantage of this straightforward method is that all sperm cells are present in the pellet including abnormal, immature, and dead spermatozoa as well as leukocytes and epithelial cells. Such unsorted samples could inhibit the capacitation process and increase the development of antisperm antibodies following insemination [86]. Moreover, oxidative damage to the sperm chromatin was reported, and since then the simple centrifugation method was no longer recommended [87].
The swim-up technique belongs to procedures where the active motion of spermatozoa plays a key role in the separation of highly motile sperm. The nutritional support is provided by antioxidants present in the culture medium. For human semen, the sample (1 mL) is gently prewashed, placed at the bottom of a 15 mL conical test tube, and covered by the sperm wash medium (1.2 mL). The tube is incubated for 60 min at an angle of 45°. The upper supernatant is sterilely collected [42]. Several modifications of swim-up technique were also reported for humans [88,89,90,91] and animal species [92,93,94]. Chen et al. [95] reported that 57.4% of semen samples processed by swim-up were bacteria-free, and only 23.8% were retrieved in the case of density gradient centrifugation. However, the most reliable centrifugation technique to date is density gradient centrifugation with subsequent swim-up. Almost 98% of the semen samples were reported to be free from bacteria.
Density gradient centrifugation is well-known in semen processing laboratories since it is a relatively simple method used in different cell separations based on density. The density of morphologically normal human sperm is 1.10 g/mL while, in the case of morphologically abnormal and immature sperm, the density is 1.06–1.09 g/mL [96]. Lindahl and Kihlström [97] reported normal bull sperm density varying between 1.241 and 1.334 g/mL. On the other hand, Lavon et al. [98] reported a range from 1.0376 to 1.0927 g/mL, because the density of sperm may change with age [99]. The basis of this separation type is a colloidal medium formed by silica particles coated with covalently bonded hydrophilic silane in HEPES [100]. Usually, two gradients are used to obtain morphologically normal motile spermatozoa with intact membranes: a denser colloid is placed at the bottom of a falcon tube and a sparser colloid is gently layered on top of the denser colloid without mixing. The common concentration and volume ratio between these phases is 1:1. The semen sample is carefully layered on top of the sparser phase and centrifuged. During centrifugation, highly motile spermatozoa reach the bottom easier than slowly moving or immotile sperm cells. After centrifugation, interphases seminal plasma/sparser colloid and sparser/denser colloid contain abnormal cells, cell debris, leukocytes, and immotile or poor motility spermatozoa. Most of the bacteria stay in the seminal plasma on top of the falcon tube. The pellet is formed by the most viable spermatozoa. Therefore, it is necessary to aseptically remove the supernatant. The pellet is washed, centrifuged, and resuspended with a washing medium again. The centrifugal force should be kept under 300× g to minimize ROS production [101,102]. To keep aseptic conditions, Nicholson et al. [102] strictly recommend changing Pasteur pipettes and tubes before washing. According to their results, approximately 30 colonies per 10 mL of semen were detected in fresh ejaculates obtained from men undergoing fertility investigation, while 3 colonies were counted after density gradient centrifugation, and less than 1 colony when pipettes and tubes were changed before washing. Single-layer centrifugation represents a modified density gradient separation when only one phase of colloid is used to separate viable spermatozoa [103]. Both single- and double-layer centrifugation can effectively improve the quality of collected semen [104]. However, the comparison of the effectivity of both methods is unknown when it comes to the elimination of bacteria. Morrell et al. [105] reported the removal of more than 90% of the bacterial load of >5 × 104 CFU/mL thanks to colloid centrifugation. What is more, higher removal effectivity was observed with lower bacterial loads. So far, human [106], bovine [103], boar [107], and equine [108] semen was successfully processed through colloid centrifugation to eliminate bacteria from samples. Despite the fact that several studies reported an improvement in the proportion of non-fragmented spermatozoa [109,110], there is probably still a certain degree of iatrogenic DNA damage because of centrifugation [111].

5. Non-Thermal Plasma

Plasma, regarded as the fourth fundamental state of matter, is defined as a quasi-neutral system in a gaseous or fluid-like form which can be artificially created in an electromagnetic field coupled with a flow of neutral gases such as oxygen, argon, helium, nitrogen, or atmospheric air [112,113]. Plasma is a heterogenous mixture composed of reactive oxygen species (hydrogen peroxide, ozone, etc.), reactive nitrogen species (RNS; nitrogen dioxide, nitric oxide, etc.), charged particles, excited atoms, electrical field, positive and negative ions, and UV radiation [114,115,116], all of which are known to possess strong antimicrobial properties. Their synergistic action results in the numerous benefits of plasma as an efficient, cost-effective, and non-toxic bactericidal agent [112].
Two plasmas exist based on the temperature and relative energy levels of electrons and particles, specifically high-temperature (thermal or fusion) plasmas and low-temperature (non-thermal or cold) plasmas [117]. As opposed to thermal plasma, non-thermal plasma (NTP) is generated at low or atmospheric pressure, containing heavy particles at room temperature and electrons at higher temperature [117,118]. Consequently, NTP application does not produce the high heat that would cause potential side-effects or harm when applied to living tissues, cells, or biomaterials [117,119].
The two most commonly used NTP configurations are the dielectric barrier discharge (DBD) and plasma jet [113] (Figure 1). The principle of DBD lies in an electrical discharge between two electrodes at atmospheric pressure and air where one electrode is supplied with a high voltage, while the other one is grounded. The high voltage electrode is enclosed within a dielectric material, such as glass or silica glass, ceramics, polymers, or quartz, whose role is to limit and/or block the discharge current [120]. Depending on the plasma configuration, the gas employed or the voltage of the DBD model, the distance between electrodes may range from micrometers to centimeters while the operating frequencies oscillate around the tens of kHz [121]. In general, DBD plasma takes advantage of atmospheric air as the working gas [113]. On the other hand, a plasma jet is a tube with electrodes placed either inside or around it, through which gas flows and ionizes once it passes through the electrical field created between the electrodes [122,123]. This plasma configuration may use a variety of working gases such as oxygen, argon, nitrogen, or helium. Unlike an arc created by the DBD plasma, jet plasma is ignited inside the tube and transported outside in a thin jet stream [113,122,123].
Depending on the objective and the plasma configuration use, three basic approaches may be used to treat samples with NTP: (1) Direct plasma treatment, which uses the target area as a counter electrode leading to homogenous plasma production with high levels of generated plasma components (DBD plasma) [124]. (2) Indirect plasma treatment, in which the plasma itself is generated between two electrodes and subsequently transported to the target area either by diffusion or a carrier gas (plasma jets) [113,124]. (3) Hybrid plasma treatment, where plasma is produced in multiple discharges such as a grounded wire mesh electrode (surface micro discharge) [113].
Non-thermal plasma has emerged as an alternative technique with exceptional potential in various biomedical directions including the sterilization and sanitation of different surfaces [114,121,125], wound healing [124,126], tissue regeneration [124,127], treatment of infections [127,128], and cancer [80,81,82]. In particular, interactions of NTP with both Gram-positive and Gram-negative bacteria, biofilms, spores, and fungi have been extensively studied over the past decade [116,129,130]. A notable antibacterial efficiency of NTP has been reported against a substantial number of high-risk pathogens such as Staphylococcus spp., Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, and Bacillus spp. in the form of liquid bacterial suspensions as well as surface-grown biofilms [131,132,133,134,135].
Nevertheless, the exact mechanism of NTP interactions with prokaryotic or eukaryotic cells has not been fully understood, which is caused by the great diversity of the biological targets, chemical, and physical properties of NTP, and above all by a significant variability of active NTP components and their subsequent effects on the target [113]. As discussed earlier, the primary components of NTP are ROS, RNS, UV radiation, and electrical field, all of which possess antimicrobial properties. More specifically, ROS and RNS act as pro-oxidants on the outer cellular structures of bacteria [129], leading to lipid peroxidation, DNA fragmentation, protein alterations, and cell death in microorganisms [136]. Charged particles and electrostatic force may equally play an important role in disintegrating the outer cell membrane [137]. Furthermore, UV radiation affects the dimerization of thymine bases in DNA, leading to irreparable damage to the nucleotides [138]. Whilst all these effects are desirable in terms of bacterial eradication, it must be remembered that the NTP treatment of semen samples may exhibit a similar negative impact on the sperm cells. As such, for NTP to be successfully applied as an antibacterial treatment option for the processing of ejaculates, pivotal reports have recently emerged studying the effects NTP on male gametes.
In the experiments designed by Mazhir et al. [139], spermatozoa collected from patients suffering asthenozoospermia were exposed to NTP discharged from a plasma jet for 120 s. The samples were compared before and following plasma treatment. The authors concluded that NTP treatment led to improved sperm motility and a lower occurrence of spermatozoa with medium and high levels of DNA damage. Inversely, the DBD plasma treatment of normozoospermic semen samples was studied by Tvrdá et al. [140]. Exposure to NTP with a power input of 40 W for 15 s or 30 s had no negative effects on the sperm motility, membrane integrity, mitochondrial activity, or DNA stability. However, a prolonged NTP treatment of 60 or 90 s impaired all target sperm quality characteristics in a time-dependent manner. The authors suggest the probable mechanism of action of high NTP doses may be connected to ROS overproduction, leading to the destabilization of the plasma membrane, lipid peroxidation, and a loss of DNA integrity. Similar observations were published by Zhang et al. [141], who subjected rooster semen to DBD plasma of different intensities for 20 s. According to the study, DBD plasma treatment of 11.7 kV led to maximum sperm motility by controlling ROS levels, upregulating the expression levels of antioxidant genes, while boosting the release of adenosine triphosphate and the activity of mitochondrial respiratory enzymes. Nevertheless, a prolonged exposure to NTP or higher plasma voltage exerted significant adverse effects on the sperm vitality and oxidative profile. Summarizing the preliminary data from the currently published studies, it may be indicated that appropriate plasma doses and exposure times need to be carefully selected to preserve sperm vitality, should NTP be used in the practical management of bacteriospermia in the future. At the same time, studies on sperm-bacterial co-cultures must be designed and carried out in order to define the optimal NTP exposure conditions for the effective eradication of bacteria while preserving the desirable sperm structural integrity and functional activity.

6. Magnetic-Activated Cell Sorting

Magnetic-activated cell sorting (MACS) is a modern sperm separation technique that has been successfully employed to eliminate apoptotic spermatozoa that may be responsible for AI or ART failure even in normozoospermic males. The magnetic separation of semen samples has been shown to yield highly motile and more cryoresistant spermatozoa with desirable viability, morphology, and fertilization potential [142,143,144,145]. The principle of the technique lies in the utilization of colloidal super-paramagnetic microbeads of approximately 50 nm, composed of a biodegradable matrix that is conjugated with annexin V [145]. This phospholipid-binding protein selectively binds to the phosphatidylserine residue that is externalized on the cellular surface if the membrane is damaged and the cell has entered apoptotic death [146]. Hence, a positive annexin V signal indicates that the sperm membrane integrity has been compromised, and the sperm cell is highly unlikely to accomplish fertilization [147].
The technique involves the incubation of the sperm suspension with annexin V-labelled microbeads at laboratory temperature for 15–20 min. The microbeads have no known effects on the sperm structure or function and, thanks to their biodegradability, it is not necessary to remove these following co-incubation [148]. The suspension is then loaded into separation columns placed within iron spheres on a stand surrounded by a magnetic field of 0.5–1.5 Tesla [100]. The resulting setting causes spermatozoa that are non-apoptotic to pass through the magnetic field, whereas sperm that are apoptotic, and thus labeled with the microbeads, are retained in the magnetic field [142,144]. Based on the labelling and binding process and subsequent magnetic separation, two sperm cell fractions will be obtained: an annexin-negative fraction, representing non-apoptotic and thus membrane-intact spermatozoa and an annexin-positive fraction, standing for apoptotic spermatozoa with an altered membrane. Following separation, the column will be removed from the MACS separator and the retained cells will be eluted with the annexin V binding buffer [148] (Figure 2).
As opposed to conventional separation methods stemming from sperm motility and/or density, MACS is based on sperm differentiation on a molecular level. The application of MACS separation in practical settings has been shown to yield highly motile spermatozoa [144] with a lower incidence of acrosomal, midpiece, and tail defects as well as cytoplasmic droplets [149,150]. Furthermore, MACS has been revealed to be highly effective in improving fertilization rates and oocyte penetration that are crucial pre-requisites for ARTs [151,152]. Consequently, MACS application has improved pregnancy rates in comparison to spermatozoa processed by density gradient alone [153]. The technique has been reported to be particularly helpful in optimizing the sperm freezing–thawing procedure by removing apoptotic spermatozoa prior to cryopreservation and hence improving the resulting post-thaw sperm cryosurvival, motility, and mitochondrial activity [148,154].
Whilst MACS is regarded as a fast, convenient, and reliable technique that is based on inexpensive and easy-to-use equipment, providing consistent results in terms of good cell purity and recovery, its major limitation currently lies in the removal of primary apoptotic spermatozoa. The technique is limited by its ability to remove other components of semen including seminal plasma, immature sperm, leukocytes, debris, and, eventually, bacteria. As such, MACS alone is not suitable for bacterial elimination, which is why it is recommended to combine the technique with other sperm-processing protocols so that these can complement each other and provide spermatozoa with the highest quality while at the same time leaving them free from any factors that may endanger their structural integrity or functional activity [144]. In fact, it has been reported that the combination of density gradient centrifugation and MACS is currently superior to other sperm preparation protocols in terms of removing immature spermatozoa, leukocytes, epithelial cells, and bacteria whilst eliminating already damaged spermatozoa with compromised membranes, activated apoptosis, oxidative damage, and DNA fragmentation [151,154].

7. Microfluidics and Microelectrophoresis

The most rapidly evolving field for sperm selection, preparation, and purification is based on microfluidics and microflow systems, considered as the semen processing methods of the 21st century [155]. The principle of microfluidics lies in the study and control of small volumes (picoliters or microliters) of fluids inside micrometer-sized channels [156] made from transparent polydimethylsiloxane silicon polymers that are nontoxic to the cells [157]. These channels are ideally suited to spermatozoa as these may be designed to resemble the architecture of the female reproductive tract, thus enabling a biomimicry-based sperm selection simulating an in vivo environment [155]. Different readily available lab-on-chip approaches may be used to purify and select spermatozoa based on motility [157,158], electrophoresis [159], flow [160], optical forces [161,162], chemical gradients [163] or chemotaxis [164,165].
Currently, the most popular devices take advantage of passive microfluidics which selects spermatozoa based on boundary-following behavior. One example uses two parallel laminar flow channels where immature and non-motile spermatozoa, leukocytes, bacteria, or debris will move along their initial stream and be collected from one outlet, while motile spermatozoa will swim into a parallel streamline and be recovered from a separate outlet [158]. Another device that has been designed for sperm processing consists of a radial network of channels containing a medium that simulates the viscosity of the female reproductive fluid, separating spermatozoa into straight, right, and left swimmers. Raw semen will be loaded into the device and kept undisturbed for 15 min. Motile spermatozoa will move through the microchannel end exit from one outlet, while immotile spermatozoa, debris, leukocytes, and bacteria will be retained in the inlet [166]. Whilst pioneer microfluid devices for semen processing relied on sperm selection based on their size or motility exclusively, a higher effectivity of the system has been achieved by incorporating chemo-attractants, such as cumulus cells, oviductal or fallopian tube cells [164,167].
Amongst the numerous advantages that come with microfluidics, the small sample volume, short processing times, increased automation, and the ability to work with single cells in a minimally invasive manner are the most prominent ones [168,169]. The yield of cells recovered by microfluid systems is comparable to other conventional methods [160], while the recovered spermatozoa present with a better progressive motility, morphology, and DNA integrity [170]. As such, microfluid platforms present with a great potential, particularly in the field of semen processing for the purposes of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in humans [170,171,172] as well as animals [173,174]. Keeping bacteria as a notable component of semen, Jeon et al. [175] have just recently proposed a fully automated multi-dimensional double spiral (MDDS) microfluidic device that can effectively isolate spermatozoa from other non-sperm seminal cells (including bacteria) in raw semen samples without any pre-washing steps. Preliminary data have revealed the excellent performance of the MDDS platform with ~80% of sperm cell recovery, and >99.95% removal of 10 μm beads serving as a surrogate for leukocytes or bacteria from both low-viscosity as well as high-viscosity ejaculates. As such, this microfluidic device could become an efficient and automated tool to collect purified sperm suspensions in the absence of any pro-inflammatory leukocytes or contaminating bacteria as an alternative to swim-up or density centrifugation, which often suffer from low sperm recovery and labor-intensive intermediate steps in the laboratory.
Finally, microelectrophoretic techniques retrieve spermatozoa from semen based on their charge and size [160,176,177]. The charge of mature spermatozoa is inherently negative [160,178], which develops with sialic acid formation on the sperm surface late in the maturation process. As such, within an electric field, more mature and negatively charged spermatozoa will be drawn towards the positively charged anode [177]. This process will enable the separation of mature and viable spermatozoa from their damaged or immature counterparts. At the same time, spermatozoa will be separated from other seminal components based on their size [160,177]. During microelectrophoresis, a polycarbonate membrane separates the electrophoretic chamber with pores large enough to enable vital spermatozoa to pass through while other larger undesirable cells in the semen will be retained on the membrane (Figure 3). A microelectrophoretic device called Felix™ that separates high quality sperm from raw semen, by a process combining electrophoresis and size exclusion membranes, is already commercially available. The device consists of a system of chambers connected with platinum-coated titanium electrodes. The sample and harvest chamber are separated by a 5 μm thick polycarbonate membrane which filters out vital and healthy spermatozoa from other contaminating cells such as germ cells, leukocytes, and, eventually, bacteria. One separation cycle lasts 6 min with a constant current of 75 mAmps and a variable voltage of 18–21 mV [160]. The device has been already tested out against conventional density gradient centrifugation across five international ART centers with both methods being capable of isolating highly vital and motile spermatozoa. Whilst the yield was lower with the Felix™ device, the recovered spermatozoa presented with a significantly improved DNA integrity relative to density gradient centrifugation. More importantly, the preparation time and workflow were significantly reduced with microelectrophoresis which may be a highly welcome asset in clinical andrology [179].

8. Conclusions

In summary, we have described selected strategies that are or may be used for the eradication of bacteria from semen while at the same time yielding spermatozoa that present with desirable viability and fertilization potential. Nevertheless, each ejaculate should be evaluated beforehand to determine the best method for bacterial decontamination depending on the initial state of the sample and its use. Future research is necessary to improve the efficiency and safety of each approach. Novel strategies to prevent bacteriospermia may improve the probability of obtaining structurally intact, viable, and mature spermatozoa without the necessity for antibiotic supplementation to semen extenders and/or ART media; nevertheless, these will still need large-scale and multicenter optimization and standardization trials before their incorporation into everyday andrological practice.

Author Contributions

Conceptualization, E.T. and M.C.; methodology, M.Ď., F.B., M.C. and E.T.; software, E.T.; formal analysis, M.Ď., F.B., M.C. and E.T.; resources, E.T.; data curation, M.Ď., F.B. and E.T.; writing—original draft preparation, M.Ď., F.B., M.C. and E.T.; writing—review and editing, E.T.; visualization, F.B.; supervision, E.T.; project administration, E.T.; funding acquisition, E.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Slovak Research and Development Agency (grant no. APVV-15-0544, APVV-21-0095) and by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic and Slovak Academy of Science (grant no. VEGA 1/0239/20).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data collected for the purposes of this paper are available upon reasonable request from the corresponding author.

Acknowledgments

We wish to thank the CeRA Team of Excellence for their support.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Hezavehei, M.; Sharafi, M.; Kouchesfahani, H.M.; Henkel, R.; Agarwal, A.; Esmaeili, V.; Shahverdi, A. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reprod. BioMed. Online 2018, 37, 327–339. [Google Scholar] [CrossRef]
  2. Sharma, Y.; Sharma, M. Sperm Cryopreservation: Principles and Biology. J. Infertil. Reprod. Biol. 2020, 8, 43–48. [Google Scholar] [CrossRef]
  3. Parekh, N.V.; Lundy, S.D.; Vij, S.C. Fertility considerations in men with testicular cancer. Transl. Androl. Urol. 2020, 9, S14–S23. [Google Scholar] [CrossRef]
  4. Tvrdá, E.; López-Fernández, C.; Sánchez-Martín, P.; Gosálvez, J. Sperm DNA fragmentation in donors and normozoospermic patients attending for a first spermiogram: Static and dynamic assessment. Andrologia 2018, 50, e12986. [Google Scholar] [CrossRef]
  5. Peris-Frau, P.; Soler, A.J.; Iniesta-Cuerda, M.; Martín-Maestro, A.; Sánchez-Ajofrín, I.; Medina-Chávez, D.A.; Fernández-Santos, M.R.; García-Álvarez, O.; Maroto-Morales, A.; Montoro, V.; et al. Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality. Int. J. Mol. Sci. 2020, 21, 2781. [Google Scholar] [CrossRef] [Green Version]
  6. Curry, M.R. Cryopreservation of semen from domestic livestock. Rev. Reprod. 2000, 5, 46–52. [Google Scholar] [CrossRef] [PubMed]
  7. Fickel, J.; Wagener, A.; Ludwig, A. Semen cryopreservation and the conservation of endangered species. Eur. J. Wildl. Res. 2007, 53, 81–89. [Google Scholar] [CrossRef]
  8. Foote, R.H. The history of artificial insemination: Selected notes and notables. J. Anim. Sci. 2002, 80, 1–10. [Google Scholar] [CrossRef] [Green Version]
  9. Rusz, A.; Pilatz, A.; Wagenlehner, F.; Linn, T.; Diemer, T.; Schuppe, H.C.; Lohmeyer, J.; Hossain, H.; Weidner, W. Influence of urogenital infections and inflammation on semen quality and male fertility. World J. Urol. 2012, 30, 23–30. [Google Scholar] [CrossRef]
  10. Pergialiotis, V.; Karampetsou, N.; Perrea, D.N.; Konstantopoulos, P.; Daskalakis, G. The impact of bacteriospermia on semen parameters: A meta-analysis. J. Fam. Reprod. Heal. 2018, 12, 73–83. [Google Scholar]
  11. Tvrdá, E.; Ďuračka, M.; Benko, F.; Lukáč, N. Bacteriospermia—A formidable player in male subfertility. Open Life Sci. 2022, 17, 1001–1029. [Google Scholar] [CrossRef] [PubMed]
  12. Fraczek, M.; Hryhorowicz, M.; Gill, K.; Zarzycka, M.; Gaczarzewicz, D.; Jedrzejczak, P.; Bilinska, B.; Piasecka, M.; Kurpisz, M. The effect of bacteriospermia and leukocytospermia on conventional and nonconventional semen parameters in healthy young normozoospermic males. J. Reprod. Immunol. 2016, 118, 18–27. [Google Scholar] [CrossRef]
  13. Tvrdá, E.; Lovíšek, D.; Gálová, E.; Schwarzová, M.; Kováčiková, E.; Kunová, S.; Žiarovská, J.; Kačániová, M. Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men. Int. J. Mol. Sci. 2022, 23, 8678. [Google Scholar] [CrossRef] [PubMed]
  14. Ďuračka, M.; Belić, L.; Tokárová, K.; Žiarovská, J.; Kačániová, M.; Lukáč, N.; Tvrdá, E. Bacterial communities in bovine ejaculates and their impact on the semen quality. Syst. Biol. Reprod. Med. 2021, 67, 438–449. [Google Scholar] [CrossRef]
  15. Tvrdá, E.; Kačániová, M.; Baláži, A.; Vašíček, J.; Vozaf, J.; Jurčík, R.; Ďuračka, M.; Žiarovská, J.; Kováč, J.; Chrenek, P. The Impact of Bacteriocenoses on Sperm Vitality, Immunological and Oxidative Characteristics of Ram Ejaculates: Does the Breed Play a Role? Animals 2022, 12, 54. [Google Scholar] [CrossRef] [PubMed]
  16. Lenický, M.; Slanina, T.; Kačániová, M.; Galovičová, L.; Petrovičová, M.; Ďuračka, M.; Benko, F.; Kováč, J.; Tvrdá, E. Identification of Bacterial Profiles and Their Interactions with Selected Quality, Oxidative, and Immunological Parameters of Turkey Semen. Animals 2021, 11, 1771. [Google Scholar] [CrossRef]
  17. Tvrdá, E.; Petrovičová, M.; Benko, F.; Ďuračka, M.; Kováč, J.; Slanina, T.; Galovičová, L.; Žiarovská, J.; Kačániová, M. Seminal Bacterioflora of Two Rooster Lines: Characterization, Antibiotic Resistance Patterns and Possible Impact on Semen Quality. Antibiotics 2023, 12, 336. [Google Scholar] [CrossRef]
  18. Gonçalves, M.F.M.; Fernandes, Â.R.; Rodrigues, A.G.; Lisboa, C. Microbiome in Male Genital Mucosa (Prepuce, Glans, and Coronal Sulcus): A Systematic Review. Microorganisms 2022, 10, 2312. [Google Scholar] [CrossRef]
  19. Zuber, A.; Peric, A.; Pluchino, N.; Baud, D.; Stojanov, M. Human Male Genital Tract Microbiota. Int. J. Mol. Sci. 2023, 24, 6939. [Google Scholar] [CrossRef]
  20. Onywera, H.; Williamson, A.L.; Ponomarenko, J.; Meiring, T.L. The Penile Microbiota in Uncircumcised and Circumcised Men: Relationships With HIV and Human Papillomavirus Infections and Cervicovaginal Microbiota. Front. Med. 2020, 7, 383. [Google Scholar] [CrossRef]
  21. Sannat, C.; Nair, A.; Sahu, S.B.; Sahasrabudhe, S.A.; Kumar, A.; Gupta, A.K.; Shende, R.K. Critical sources of bacterial contamination and adoption of standard sanitary protocol during semen collection and processing in Semen Station. Vet. World 2015, 8, 631–635. [Google Scholar] [CrossRef] [PubMed]
  22. Lin, L.L.; Guu, H.F.; Yi, Y.C.; Kung, H.F.; Chang, J.C.; Chen, Y.F.; Chen, L.Y.; Chen, M.J. Contamination of ART culture Media-The role of semen and strategies for prevention. Taiwan. J. Obstet. Gynecol. 2021, 60, 523–525. [Google Scholar] [CrossRef]
  23. Bustani, G.S.; Baiee, F.H. Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders. Vet. World 2021, 14, 1220–1233. [Google Scholar] [CrossRef] [PubMed]
  24. Morrell, J.M.; Wallgren, M. Alternatives to antibiotics in semen extenders: A review. Pathogens 2014, 3, 934–946. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Nedwell, D.B. Effect of low temperature on microbial growth: Lowered affinity for substrates limits growth at low temperature. FEMS Microbiol. Ecol. 1999, 30, 101–111. [Google Scholar] [CrossRef]
  26. Levy, R.; Grattard, F.; Maubon, I.; Ros, A.; Pozzetto, B. Bacterial risk and sperm cryopreservation. Andrologia 2004, 36, 282–285. [Google Scholar] [CrossRef]
  27. Eini, F.; Kutenaei, M.A.; Zareei, F.; Dastjerdi, Z.S.; Shirzeyli, M.H.; Salehi, E. Effect of bacterial infection on sperm quality and DNA fragmentation in subfertile men with leukocytospermia. BMC Mol. Cell. Biol. 2021, 22, 42. [Google Scholar] [CrossRef]
  28. Gholami, M.; Moosazadeh, M.; Haghshenash, M.R.; Jafarpour, H.; Mousavi, T. Evaluation of the Presence of Bacterial and Viral Agents in the Semen of Infertile Men: A Systematic and Meta-Analysis Review Study. Front. Med. 2022, 9, 835254. [Google Scholar] [CrossRef]
  29. Rennemeier, C.; Frambach, T.; Hennicke, F.; Dietl, J.; Staib, P. Microbial quorum-sensing molecules induce acrosome loss and cell death in human spermatozoa. Infect. Immun. 2009, 77, 4990–4997. [Google Scholar] [CrossRef] [Green Version]
  30. Fraczek, M.; Piasecka, M.; Gaczarzewicz, D.; Szumala-Kakol, A.; Kazienko, A.; Lenart, S.; Laszczynska, M.; Kurpisz, M. Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus. Andrologia 2012, 44, 315–329. [Google Scholar] [CrossRef]
  31. Fraczek, M.; Kurpisz, M. Mechanisms of the harmful effects of bacterial semen infection on ejaculated human spermatozoa: Potential inflammatory markers in semen. Folia Histochem. Cytobiol. 2015, 53, 201–217. [Google Scholar] [CrossRef] [Green Version]
  32. Tvrdá, E.; Bučko, O.; Rojková, K.; Ďuračka, M.; Kunová, S.; Kováč, J.; Benko, F.; Kačániová, M. The Efficiency of Selected Extenders against Bacterial Contamination of Boar Semen in a Swine Breeding Facility in Western Slovakia. Animals 2021, 11, 3320. [Google Scholar] [CrossRef] [PubMed]
  33. Givens, M.D. Review: Risks of disease transmission through semen in cattle. Animal 2018, 12, s165–s171. [Google Scholar] [CrossRef] [Green Version]
  34. Samper, J.C.; Tibary, A. Disease transmission in horses. Theriogenology 2006, 66, 551–559. [Google Scholar] [CrossRef] [PubMed]
  35. Martins Pereira, E.C.; Silva, A.; da Costa, E.P.; Real Pereir, C.E. Success in Artificial Insemination, 1st ed.; Lemma, A., Ed.; InTech: London, UK, 2013; pp. 117–139. [Google Scholar]
  36. Bielanski, A.; Bergeron, H.; Lau, P.C.; Devenish, J. Microbial contamination of embryos and semen during long term banking in liquid nitrogen. Cryobiology 2003, 46, 146–152. [Google Scholar] [CrossRef] [PubMed]
  37. Krissi, H.; Orvieto, R.; Ashkenazi, J.; Gilboa, Y.; Shalev, J.; Moscovitch, I.; Bar-Hava, I. Effect of contaminated preprocessed semen on fertilization rate and embryo quality in assisted reproductive techniques. Gynecol. Endocrinol. 2004, 18, 63–67. [Google Scholar] [CrossRef] [PubMed]
  38. Flasche, S.; Atkins, K.E. Balancing Benefits and Risks of Antibiotic Use. J. Infect. Dis. 2018, 218, 1351–1353. [Google Scholar] [CrossRef] [Green Version]
  39. Council Directive 88/407/EEC of 14 June 1988 Laying Down the Animal Health Requirements Applicable to Intra-Community Trade in and Imports of Deep-Frozen Semen of Domestic Animals of the Bovine Species. Available online: https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX%3A31988L0407 (accessed on 15 June 2023).
  40. Council Directive 90/429/EEC of 26 June 1990 Laying Down the Animal Health Requirements Applicable to Intra-Community Trade in and Imports of Semen of Domestic Animals of the Porcine Species. Available online: https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex:31990L0429 (accessed on 15 June 2023).
  41. Council Directive 92/65/EEC of 13 July 1992 Laying Down Animal Health Requirements Governing Trade in and Imports Into the Community of Animals, Semen, Ova and Embryos not Subject to Animal Health Requirements Laid Down in Specific Community Rules Referred to in Annex A (I) to Directive 90/425/EEC. Available online: https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:31992L0065 (accessed on 15 June 2023).
  42. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen, 6th ed.; WHO Press: Geneva, Switzerland, 2021; 276p. [Google Scholar]
  43. Santos, C.S.; Silva, A.R. Current and alternative trends in antibacterial agents used in mammalian semen technology. Anim. Reprod. 2020, 17, e20190111. [Google Scholar] [CrossRef] [Green Version]
  44. Ventola, C.L. The antibiotic resistance crisis: Part 1: Causes and threats. Pharm. Ther. 2015, 40, 277–283. [Google Scholar]
  45. Santos, C.S.; Campos, L.B.; Praxedes, É.C.G.; Moreira, S.S.J.; Souza-Júnior, J.B.F.; Comizzoli, P.; Feijó, F.M.C.; Silva, A.R. Influence of antibiotics on bacterial load and sperm parameters during short-term preservation of collared peccary semen. Anim. Reprod. 2021, 18, e20210021. [Google Scholar] [CrossRef]
  46. Tímermans, A.; Vázquez, R.; Otero, F.; Gosálvez, J.; Johnston, S.; Fernández, J.L. Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay. Andrologia 2022, 54, e14328. [Google Scholar] [CrossRef] [PubMed]
  47. Anel-Lopez, L.; Riesco, M.F.; Montes-Garrido, R.; Neila-Montero, M.; Boixo, J.C.; Chamorro, C.; Ortega-Ferrusola, C.; Carvajal, A.; Altonaga, J.R.; de Paz, P.; et al. Comparing the Effect of Different Antibiotics in Frozen-Thawed Ram Sperm: Is It Possible to Avoid Their Addition? Front. Vet. Sci. 2021, 8, 656937. [Google Scholar] [CrossRef]
  48. Olana, S.; Mazzilli, R.; Santino, I.; Martinelli, D.; Zamponi, V.; Macera, M.; Salerno, G.; Mazzilli, F.; Faggiano, A.; Gianfrilli, D. Sperm culture and bacterial susceptibility to antibiotics in a large andrological population: Prevalence and impact on seminal parameters. Int. Microbiol. 2023, 26, 69–79. [Google Scholar] [CrossRef] [PubMed]
  49. Faisal, A.J.; Salman, H.A. Determination of Semen Quality and Antibacterial Susceptibility Pattern of Bacteria Isolated from Semen of Iraqi Subjects. Microbiol. Biotechnol. Lett. 2021, 49, 587–593. [Google Scholar] [CrossRef]
  50. Al-Jebouri, M.M.; Mdish, S.A. Tracing of antibiotic-resistant bacteria isolated from semen of Iraqi males with primary infertility. Open J. Urol. 2019, 9, 19–29. [Google Scholar] [CrossRef] [Green Version]
  51. Isaiah, I.N.; Nche, B.T.; Nwagu, I.G.; Nnanna, I.I. Current studies on bacteriospermia the leading cause of male infertility: A protégé and potential threat towards mans extinction. N. Am. J. Med. Sci. 2011, 3, 562–564. [Google Scholar] [CrossRef]
  52. Machen, G.L.; Bird, E.T.; Brown, M.L.; Ingalsbe, D.A.; East, M.M.; Reyes, M.; Kuehl, T.J. Time trends for bacterial species and resistance patterns in semen in patients undergoing evaluation for male infertility. Bayl. Univ. Med. Cent. Proc. 2018, 31, 165–167. [Google Scholar] [CrossRef] [Green Version]
  53. Goularte, K.L.; Voloski, F.L.S.; Redú, J.F.M.; Ferreira, C.E.R.; Vieira, A.D.; Duval, E.H.; Mondadori, R.G.; Lucia, T., Jr. Antibiotic resistance in microorganisms isolated in a bull semen stud. Reprod. Domest. Anim. 2020, 55, 318–324. [Google Scholar] [CrossRef] [PubMed]
  54. Webb, E.M.; Holman, D.B.; Schmidt, K.N.; Crouse, M.S.; Dahlen, C.R.; Cushman, R.A.; Snider, A.P.; McCarthy, K.L.; Amat, S. A Longitudinal Characterization of the Seminal Microbiota and Antibiotic Resistance in Yearling Beef Bulls Subjected to Different Rates of Gain. Microbiol. Spectr. 2023, 11, e0518022. [Google Scholar] [CrossRef]
  55. Yániz, J.L.; Marco-Aguado, M.A.; Mateos, J.A.; Santolaria, P. Bacterial contamination of ram semen, antibiotic sensitivities, and effects on sperm quality during storage at 15 °C. Anim. Reprod. Sci. 2010, 122, 142–149. [Google Scholar] [CrossRef]
  56. Costinar, L.; Herman, V.; Pitoiu, E.; Iancu, I.; Degi, J.; Hulea, A.; Pascu, C. Boar Semen Contamination: Identification of Gram-Negative Bacteria and Antimicrobial Resistance Profile. Animals 2021, 12, 43. [Google Scholar] [CrossRef] [PubMed]
  57. Bresciani, C.; Cabassi, C.S.; Morini, G.; Taddei, S.; Bettini, R.; Bigliardi, E.; Di Ianni, F.; Sabbioni, A.; Parmigiani, E. Boar Semen Bacterial Contamination in Italy and Antibiotic Efficacy in a Modified Extender. Ital. J. Anim. Sci. 2014, 13, 3082. [Google Scholar] [CrossRef] [Green Version]
  58. Alkali, I.M.; Asuku, S.O.; Umar, M.B.; Abba, A.; Mustapha, A.; Bukar, M.M.; Waziri, M.A. Microbial Contaminants in Fresh and Extended Turkey Semen and their Sensitivity to Antibiotics. Nig. Vet. J. 2020, 41, 1–6. [Google Scholar] [CrossRef]
  59. Maasjost, J.; Mühldorfer, K.; de Jäckel, S.C.; Hafez, H.M. Antimicrobial Susceptibility Patterns of Enterococcus faecalis and Enterococcus faecium Isolated from Poultry Flocks in Germany. Avian Dis. 2015, 59, 143–148. [Google Scholar] [CrossRef]
  60. Rakha, B.A.; Zafar, Z.; Ansari, M.S.; Akhter, S.; Qadeer, S.; Akhter, A.; Waseem, K.; Santiago-Moreno, J. Influence of Bacterial Contamination and Antibiotic Sensitivity on Cryopreserved Sperm Quality of Indian Red Jungle Fowl. Biopreservation Biobanking 2023. Advance online publication. [Google Scholar] [CrossRef]
  61. Zhao, X.; Wu, H.; Lu, H.; Li, G.; Huang, Q. LAMP: A database linking antimicrobial peptides. PLoS ONE 2013, 8, e66557. [Google Scholar] [CrossRef] [Green Version]
  62. Epand, R.M.; Vogel, H.J. Diversity of antimicrobial peptides and their mechanisms of action. Biochim. Biophys. Acta. 1999, 1462, 11–28. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 2002, 415, 389–395. [Google Scholar] [CrossRef]
  64. Hensel, B.; Jakop, U.; Scheinpflug, K.; Mühldorfer, K.; Schröter, F.; Schäfer, J.; Greber, K.; Jung, M.; Schulze, M. Low temperature preservation of porcine semen: Influence of short antimicrobial lipopeptides on sperm quality and bacterial load. Sci. Rep. 2020, 10, 13225. [Google Scholar] [CrossRef]
  65. Brender, J.R.; McHenry, A.J.; Ramamoorthy, A. Does cholesterol play a role in the bacterial selectivity of antimicrobial peptides? Front. Immunol. 2012, 3, 195. [Google Scholar] [CrossRef] [Green Version]
  66. Bussalleu, E.; Sancho, S.; Briz, M.D.; Yeste, M.; Bonet, S. Do antimicrobial peptides PR-39, PMAP-36 and PMAP-37 have any effect on bacterial growth and quality of liquid-stored boar semen? Theriogenology 2017, 89, 235–243. [Google Scholar] [CrossRef] [PubMed]
  67. Schulze, M.; Dathe, M.; Waberski, D.; Müller, K. Liquid storage of boar semen: Current and future perspectives on the use of cationic antimicrobial peptides to replace antibiotics in semen extenders. Theriogenology 2016, 85, 39–46. [Google Scholar] [CrossRef] [PubMed]
  68. Sarkar, T.; Chetia, M.; Chatterjee, S. Antimicrobial peptides and proteins: From nature’s reservoir to the laboratory and beyond. Front. Chem. 2021, 9, 691532. [Google Scholar] [CrossRef] [PubMed]
  69. Moretti, E.; Signorini, C.; Corsaro, R.; Giamalidi, M.; Collodel, G. Human Sperm as an In Vitro Model to Assess the Efficacy of Antioxidant Supplements during Sperm Handling: A Narrative Review. Antioxidants 2023, 12, 1098. [Google Scholar] [CrossRef] [PubMed]
  70. Tvrdá, E.; Debacker, M.; Ďuračka, M.; Kováč, J.; Bučko, O. Quercetin and Naringenin Provide Functional and Antioxidant Protection to Stored Boar Semen. Animals 2020, 10, 1930. [Google Scholar] [CrossRef] [PubMed]
  71. Baňas, Š.; Benko, F.; Ďuračka, M.; Lukáč, N.; Tvrdá, E. Epicatechin Prevents Cryocapacitation of Bovine Spermatozoa through Antioxidant Activity and Stabilization of Transmembrane Ion Channels. Int. J. Mol. Sci. 2023, 24, 2510. [Google Scholar] [CrossRef] [PubMed]
  72. Wang, Y.; Zhang, L.; Sohail, T.; Kang, Y.; Sun, X.; Li, Y. Chlorogenic Acid Improves Quality of Chilled Ram Sperm by Mitigating Oxidative Stress. Animals 2022, 12, 163. [Google Scholar] [CrossRef]
  73. Tvrdá, E.; Petrovičová, M.; Benko, F.; Ďuračka, M.; Galovičová, L.; Slanina, T.; Kačániová, M. Curcumin Attenuates Damage to Rooster Spermatozoa Exposed to Selected Uropathogens. Pharmaceutics 2023, 15, 65. [Google Scholar] [CrossRef]
  74. Shamsudin, N.F.; Ahmed, Q.U.; Mahmood, S.; Ali Shah, S.A.; Khatib, A.; Mukhtar, S.; Alsharif, M.A.; Parveen, H.; Zakaria, Z.A. Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation. Molecules 2022, 27, 1149. [Google Scholar] [CrossRef]
  75. Cushnie, T.P.T.; Lamb, A.J. Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents 2005, 26, 343–356. [Google Scholar] [CrossRef]
  76. Górniak, I.; Bartoszewski, R.; Króliczewski, J. Comprehensive review of antimicrobial activities of plant flavonoids. Phytochem. Rev. 2019, 18, 241–272. [Google Scholar] [CrossRef] [Green Version]
  77. Mazurova, J.; Kukla, R.; Rozkot, M.; Lustykova, A.; Slehova, E.; Sleha, R.; Opletal, L. Use of natural substances for boar semen decontamination. Vet. Med. 2015, 60, 235–247. [Google Scholar] [CrossRef] [Green Version]
  78. Kou, Z.; Wang, C.; Gao, L.; Chu, G.; Yang, G.; Pang, W. Icariin improves pig sperm quality through antioxidant and antibacterial effects during liquid storage at 17 C. Livestock Sci. 2022, 256, 104827. [Google Scholar] [CrossRef]
  79. Cojkic, A.; Hansson, I.; Johannisson, A.; Morrell, J.M. Effect of Some Plant-Based Substances on Microbial Content and Sperm Quality Parameters of Bull Semen. Int. J. Mol. Sci. 2023, 24, 3435. [Google Scholar] [CrossRef]
  80. Elmi, A.; Prosperi, A.; Zannoni, A.; Bertocchi, M.; Scorpio, D.G.; Forni, M.; Foni, E.; Bacci, M.L.; Ventrella, D. Antimicrobial capabilities of non-spermicidal concentrations of tea tree (Melaleuca alternifolia) and rosemary (Rosmarinus officinalis) essential oils on the liquid phase of refrigerated swine seminal doses. Res. Vet. Sci. 2019, 127, 76–81. [Google Scholar] [CrossRef] [PubMed]
  81. Tsakmakidis, I.A.; Samaras, T.; Anastasiadou, S.; Basioura, A.; Ntemka, A.; Michos, I.; Simeonidis, K.; Karagiannis, I.; Tsousis, G.; Angelakeris, M.; et al. Iron Oxide Nanoparticles as an Alternative to Antibiotics Additive on Extended Boar Semen. Nanomaterials 2020, 10, 1568. [Google Scholar] [CrossRef] [PubMed]
  82. Tsakmakidis, I.A.; Samaras, T.; Anastasiadou, S.; Basioura, A.; Ntemka, A.; Michos, I.; Simeonidis, K.; Karagiannis, I.; Tsousis, G.; Angelakeris, M.; et al. Toxic and Microbiological Effects of Iron Oxide and Silver Nanoparticles as Additives on Extended Ram Semen. Animals 2021, 11, 1011. [Google Scholar] [CrossRef]
  83. Yousef, M.I.; Abd, H.H.; Helmy, Y.M.; Kamel, M.A. Synergistic effect of curcumin and chitosan nanoparticles on nano-hydroxyapatite-induced reproductive toxicity in rats. Environ. Sci. Pollut. Res. Int. 2021, 28, 9362–9376. [Google Scholar] [CrossRef]
  84. Viudes-de-Castro, M.P.; Marco-Jimenez, F.; Vicente, J.S.; Marin, C. Antibacterial Activity of Some Molecules Added to Rabbit Semen Extender as Alternative to Antibiotics. Animals 2021, 11, 1178. [Google Scholar] [CrossRef]
  85. Björndahl, L.; Mortimer, D.; Barratt, C.; Castilla, J.; Menkveld, R.; Kvist, U.; Alvarez, J.G.; Haugen, T. A Practical Guide to Basic Laboratory Andrology, 1st ed.; Cambridge University Press: Cambridge, UK, 2010; p. 336. [Google Scholar]
  86. Friedman, A.J.; Juneau-Norcross, M.; Sedensky, B. Antisperm antibody production following intrauterine insemination. Hum. Reprod. 1991, 6, 1125–1128. [Google Scholar] [CrossRef]
  87. Mortimer, D. Sperm preparation techniques and iatrogenic failures of in-vitro fertilization. Hum. Reprod. 1991, 6, 173–176. [Google Scholar] [CrossRef] [PubMed]
  88. Mahadevan, M.; Baker, G. Assessment and Preparation of Semen for In Vitro Fertilization. In Clinical In Vitro Fertilization, 1st ed.; Wood, C., Trounson, A., Eds.; Springer London: London, UK, 1984; pp. 83–97. [Google Scholar] [CrossRef]
  89. Henkel, R.R.; Schill, W.B. Sperm preparation for ART. Reprod. Biol. Endocrinol. 2003, 1, 108. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  90. Al Hasani, S.; Küpker, W.; Baschat, A.A.; Sturm, R.; Bauer, O.; Diedrich, C.; Diedrich, K. Mini-swim-up: A new technique of sperm preparation for intracytoplasmic sperm injection. J. Assist. Reprod. Genet. 1995, 12, 428–433. [Google Scholar] [CrossRef]
  91. Volpes, A.; Sammartano, F.; Rizzari, S.; Gullo, S.; Marino, A.; Allegra, A. The pellet swim-up is the best technique for sperm preparation during in vitro fertilization procedures. J. Assist. Reprod. Genet. 2016, 33, 765–770. [Google Scholar] [CrossRef]
  92. Arias, M.E.; Andara, K.; Briones, E.; Felmer, R. Bovine sperm separation by Swim-up and density gradients (Percoll and BoviPure): Effect on sperm quality, function and gene expression. Reprod Biol. 2017, 17, 126–132. [Google Scholar] [CrossRef]
  93. Magdanz, V.; Boryshpolets, S.; Ridzewski, C.; Eckel, B.; Reinhardt, K. The motility-based swim-up technique separates bull sperm based on differences in metabolic rates and tail length. PLoS ONE 2019, 10, e0223576. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  94. Alves, N.C.; Diniz, S.A.; Viegas, R.N.; Arigoni, A.L.; Freitas, M.M.; Lana, Â.Q.; Lagares, M.A. Action of swim-up and caffeine on equine frozen sperm. Anim. Reprod. 2022, 19, e20220056. [Google Scholar] [CrossRef] [PubMed]
  95. Chen, Z.; Jiang, L.; Wang, H.; Quan, W.; Yang, L.; Ou, C.; Sun, L. Elimination of bacteria from semen using a combination of density gradient centrifugation and swim-up. J. Men’s Health 2022, 18, 124. [Google Scholar] [CrossRef]
  96. Oshio, S.; Kaneko, S.; Iizuka, R.; Mohri, H. Effects of gradient centrifugation on human sperm. Arch. Androl. 1987, 19, 85–93. [Google Scholar] [CrossRef]
  97. Lindahl, P.E.; Kihlström, J.E. Alterations in specific gravity during ripening of bull spermatozoa. J. Dairy. Sci. 1952, 35, 393–402. [Google Scholar] [CrossRef]
  98. Lavon, U.; Volcani, R.; Danon, D. The specific gravity of bull spermatozoa. J. Reprod. Fert. 1966, 11, 447–449. [Google Scholar] [CrossRef] [Green Version]
  99. Hough, S.R.; Foote, R.H. Effect of density gradient osmolality on specific gravity of bull sperm and their separation, but not on the basis of sex. J. Rep. Develop. 2002, 48, 399–407. [Google Scholar] [CrossRef]
  100. Sharma, R.; Agarwal, A. Sperm Processing and Selection. In Male Infertility, 1st ed.; Parekattil, S., Esteves, S., Agarwal, A., Eds.; Springer: Cham, Germany, 2020; pp. 647–659. [Google Scholar]
  101. Ali, A.H.; Ajina, T.; Ali, M.B.; Mehdi, M. Efficacy of density gradient centrifugation technique (DGC) in enhancing sperm cell DNA quality for assisted reproductive technique. Middle East Fertil. Soc. J. 2022, 27, 22. [Google Scholar] [CrossRef]
  102. Nicholson, C.M.; Abramsson, L.; Holm, S.E.; Bjurulf, E. Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane-coated silica particles at different g forces. Hum. Reprod. 2000, 15, 662–666. [Google Scholar] [CrossRef] [Green Version]
  103. Ďuračka, M.; Husarčíková, K.; Jančov, M.; Galovičová, L.; Kačániová, M.; Lukáč, N.; Tvrdá, E. Staphylococcus-Induced Bacteriospermia In Vitro: Consequences on the Bovine Spermatozoa Quality, Extracellular Calcium and Magnesium Content. Animals 2021, 11, 3309. [Google Scholar] [CrossRef] [PubMed]
  104. Gloria, A.; Carluccio, A.; Wegher, L.; Robbe, D.; Befacchia, G.; Contri, A. Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates. J. Anim. Sci. Biotechnol. 2016, 7, 30. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  105. Morrell, J.M.; Klein, C.; Lundeheim, N.; Erol, E.; Troedsson, M.H.T. Removal of bacteria from stallion semen by colloid centrifugation. Anim. Reprod. Sci. 2014, 145, 47–53. [Google Scholar] [CrossRef]
  106. Fourie, J.; Loskutoff, N.; Huyser, C. Elimination of bacteria from human semen during sperm preparation using density gradient centrifugation with a novel tube insert. Andrologia 2012, 44, 513–517. [Google Scholar] [CrossRef] [Green Version]
  107. Morrell, J.M.; Wallgren, M. Colloid centrifugation of boar semen. Reprod. Domest. Anim. 2011, 46, 18–22. [Google Scholar] [CrossRef]
  108. Guimarães, T.; Lopes, G.; Pinto, M.; Silva, E.; Miranda, C.; Correia, M.J.; Damásio, L.; Thompson, G.; Rocha, A. Colloid centrifugation of fresh stallion semen before cryopreservation decreased microorganism load of frozen-thawed semen without affecting seminal kinetics. Theriogenology 2015, 83, 186–191. [Google Scholar] [CrossRef]
  109. Crespo, F.; Gutiérrez-Cepeda, L.; Gosalvez, J.; Serres, C.; Johnston, S.D. Colloidal centrifugation of stallion semen results in a reduced rate of sperm DNA fragmentation. Reprod. Domest. Anim. 2013, 48, e23–e25. [Google Scholar] [CrossRef] [PubMed]
  110. Muratori, M.; Tarozzi, N.; Carpentiero, F.; Danti, S.; Perrone, F.M.; Cambi, M.; Casini, A.; Azzari, C.; Boni, L.; Maggi, M.; et al. Sperm selection with density gradient centrifugation and swim up: Effect on DNA fragmentation in viable spermatozoa. Sci. Rep. 2019, 9, 7492. [Google Scholar] [CrossRef] [Green Version]
  111. Zini, A.; Finelli, A.; Phang, D.; Jarvi, K. Influence of semen processing technique on human sperm DNA integrity. Urology 2000, 56, 1081–1084. [Google Scholar] [CrossRef]
  112. Todorova, Y.; Benova, E.; Marinova, P.; Yotinov, I.; Bogdanov, T.; Topalova, Y. Non-Thermal Atmospheric Plasma for Microbial Decontamination and Removal of Hazardous Chemicals: An Overview in the Circular Economy Context with Data for Test Applications of Microwave Plasma Torch. Processes 2022, 10, 554. [Google Scholar] [CrossRef]
  113. Gupta, T.T.; Ayan, H. Application of Non-Thermal Plasma on Biofilm: A Review. Appl. Sci. 2019, 9, 3548. [Google Scholar] [CrossRef] [Green Version]
  114. Marinova, P.; Benova, E.; Todorova, Y.; Topalova, Y.; Yotinov, I.; Atanasova, M.; Krcma, F. Surface-wave-sustained plasma torch for water treatment. J. Phys. Conf. Ser. 2018, 982, 012009. [Google Scholar] [CrossRef]
  115. Jha, N.; Ryu, J.J.; Choi, E.H.; Kaushik, N.K. Generation and role of reactive oxygen and nitrogen species induced by plasma, lasers, chemical agents, and other systems in dentistry. Oxid. Med. Cell. Longev. 2017, 2017, 7542540. [Google Scholar] [CrossRef] [PubMed]
  116. Graves, D.B. The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology. J. Phys. D Appl. Phys. 2012, 45, 263001. [Google Scholar] [CrossRef]
  117. Hippler, R.; Kersten, H.; Schmidt, M.; Schoenbach, K.H. Low Temperature Plasmas: Fundamentals, Technologies and Techniques, 2nd ed.; Wiley-VCH: Berlin, Germany, 2008; p. 945. [Google Scholar]
  118. Stoffels, E.; Sakiyama, Y.; Graves, D.B. Cold atmospheric plasma: Charged species and their interactions with cells and tissues. IEEE Trans. Plasma Sci. 2008, 36, 1441–1457. [Google Scholar] [CrossRef]
  119. Laroussi, M. Low-Temperature Plasmas for Medicine? IEEE Trans. Plasma Sci. 2009, 37, 714–725. [Google Scholar] [CrossRef]
  120. Kogelschatz, U. Dielectric-barrier discharges: Their history, discharge physics, and industrial applications. Plasma Chem. Plasma Process. 2003, 23, 1–46. [Google Scholar] [CrossRef]
  121. Ehlbeck, J.; Schnabel, U.; Polak, M.; Winter, J.; Von Woedtke, T.; Brandenburg, R.; Von dem Hagen, T.; Weltmann, K.D. Low temperature atmospheric pressure plasma sources for microbial decontamination. J. Phys. D Appl. Phys. 2010, 44, 13002. [Google Scholar] [CrossRef] [Green Version]
  122. Kogelschatz, U. Atmospheric-pressure plasma technology. Plasma Phys. Control. Fusion 2004, 46, B63. [Google Scholar] [CrossRef]
  123. Winter, J.; Brandenburg, R.; Weltmann, K.D. Atmospheric pressure plasma jets: An overview of devices and new directions. Plasma Sources Sci. Technol. 2015, 24, 64001. [Google Scholar] [CrossRef]
  124. Isbary, G.; Shimizu, T.; Li, Y.-F.; Stolz, W.; Thomas, H.M.; Morfill, G.E.; Zimmermann, J.L. Cold atmospheric plasma devices for medical issues. Expert Rev. Med. Devices 2013, 10, 367–377. [Google Scholar] [CrossRef]
  125. Ayan, H.; Staack, D.; Fridman, G.; Gutsol, A.; Mukhin, Y.; Starikovskii, A.; Fridman, A.; Friedman, G. Application of nanosecond-pulsed dielectric barrier discharge for biomedical treatment of topographically non-uniform surfaces. J. Phys. D Appl. Phys. 2009, 42, 125202. [Google Scholar] [CrossRef]
  126. Isbary, G.; Stolz, W.; Shimizu, T.; Monetti, R.; Bunk, W.; Schmidt, H.-U.; Morfill, G.E.; Klämpfl, T.G.; Steffes, B.; Thomas, H.M. Cold atmospheric argon plasma treatment may accelerate wound healing in chronic wounds: Results of an open retrospective randomized controlled study in vivo. Clin. Plasma Med. 2013, 1, 25–30. [Google Scholar] [CrossRef]
  127. Kong, M.G.; Kroesen, G.; Morfill, G.; Nosenko, T.; Shimizu, T.; Van Dijk, J.; Zimmermann, J.L. Plasma medicine: An introductory review. New J. Phys. 2009, 11, 115012. [Google Scholar] [CrossRef]
  128. Fridman, G.; Friedman, G.; Gutsol, A.; Shekhter, A.B.; Vasilets, V.N.; Fridman, A. Applied plasma medicine. Plasma Process. Polym. 2008, 5, 503–533. [Google Scholar] [CrossRef]
  129. Moreau, M.; Orange, N.; Feuilloley, M.G.J. Non-thermal plasma technologies: New tools for bio-decontamination. Biotechnol. Adv. 2008, 26, 610–617. [Google Scholar] [CrossRef]
  130. Hoffmann, C.; Berganza, C.; Zhang, J. Cold Atmospheric Plasma: Methods of production and application in dentistry and oncology. Med. Gas Res. 2013, 3, 21. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  131. Pan, J.; Sun, K.; Liang, Y.; Sun, P.; Yang, X.; Wang, J.; Zhang, J.; Zhu, W.; Fang, J.; Becker, K.H. Cold plasma therapy of a tooth root canal infected with Enterococcus faecalis biofilms in vitro. J. Endod. 2013, 39, 105–110. [Google Scholar] [CrossRef]
  132. Molnar, I.; Papp, J.; Simon, A.; Anghel, S.D. Deactivation of Streptococcus mutans biofilms on a tooth surface using the dielectric barrier discharge at atmospheric pressure. Plasma Sci. Technol. 2013, 15, 535. [Google Scholar] [CrossRef] [Green Version]
  133. Marchal, F.; Robert, H.; Merbahi, N.; Fontagne-Faucher, C.; Yousfi, M.; Romain, C.E.; Eichwald, O.; Rondel, C.; Gabriel, B. Inactivation of Gram-positive biofilms by low-temperature plasma jet at atmospheric pressure. J. Phys. D Appl. Phys. 2012, 45, 345202. [Google Scholar] [CrossRef]
  134. Joshi, S.G.; Paff, M.; Friedman, G.; Fridman, G.; Fridman, A.; Brooks, A.D. Control of methicillin-resistant Staphylococcus aureus in planktonic form and biofilms: A biocidal efficacy study of nonthermal dielectric-barrier discharge plasma. Am. J. Infect. Control 2010, 38, 293–301. [Google Scholar] [CrossRef]
  135. Ziuzina, D.; Patil, S.; Cullen, P.J.; Boehm, D.; Bourke, P. Dielectric barrier discharge atmospheric cold plasma for inactivation of Pseudomonas aeruginosa biofilms. Plasma Med. 2014, 4, 137–152. [Google Scholar] [CrossRef] [Green Version]
  136. Liao, X.; Liu, D.; Xiang, Q.; Ahn, J.; Chen, S.; Ye, X.; Ding, T. Inactivation mechanisms of non-thermal plasma on microbes: A review. Food Control 2017, 75, 83–91. [Google Scholar] [CrossRef]
  137. Laroussi, M. Low temperature plasma-based sterilization: Overview and state-of-the-art. Plasma Process. Polym. 2005, 2, 391–400. [Google Scholar] [CrossRef]
  138. Maisch, T.; Shimizu, T.; Isbary, G.; Heinlin, J.; Karrer, S.; Klämpfl, T.G.; Li, Y.-F.; Morfill, G.; Zimmermann, J.L. Contact-free inactivation of Candida albicans biofilms by cold atmospheric air plasma. Appl. Environ. Microbiol. 2012, 78, 4242–4247. [Google Scholar] [CrossRef] [Green Version]
  139. Mazhir, S.N.; Al-Ahmed, H.I.; Hanon, M.S.; Ali, A.H. Influence of cold plasma on sperm motility and oxidative stress in patient with asthenospermia. Biosci. Res. 2019, 16, 480–484. [Google Scholar] [CrossRef]
  140. Tvrdá, E.; Lovíšek, D.; Kyzek, S.; Kováčik, D.; Gálová, E. The Effect of Non-Thermal Plasma on the Structural and Functional Characteristics of Human Spermatozoa. Int. J. Mol. Sci. 2021, 22, 4979. [Google Scholar] [CrossRef]
  141. Zhang, J.J.; Do, H.L.; Chandimali, N.; Lee, S.B.; Mok, Y.S.; Kim, N.; Kim, S.B.; Kwon, T.; Jeong, D.K. Non-thermal plasma treatment improves chicken sperm motility via the regulation of demethylation levels. Sci. Rep. 2018, 8, 7576. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  142. Grunewald, S.; Paasch, U.; Glander, H.J. Enrichment of non-apoptotic human spermatozoa after cryopreservation by immunomagnetic cell sorting. Cell Tissue Bank. 2001, 2, 127–133. [Google Scholar] [CrossRef] [PubMed]
  143. Grunewald, S.; Paasch, U.; Said, T.M.; Rasch, M.; Agarwal, A.; Glander, H.J. Magnetic-activated cell sorting before cryopreservation preserves mitochondrial integrity in human spermatozoa. Cell Tissue Bank. 2006, 7, 99–104. [Google Scholar] [CrossRef]
  144. Said, T.M.; Grunewald, S.; Paasch, U.; Glander, H.J.; Baumann, T.; Kriegel, C.; Li, L.; Agarwal, A. Advantage of combining magnetic cell separation with sperm preparation techniques. Reprod. BioMed. Online 2005, 10, 740–746. [Google Scholar] [CrossRef] [PubMed]
  145. Gil, M.; Sar-Shalom, V.; Melendez Sivira, Y.; Carreras, R.; Checa, M.A. Sperm selection using magnetic activated cell sorting (MACS) in assisted reproduction: A systematic review and meta-analysis. J. Assist. Reprod. Genet. 2013, 30, 479–485. [Google Scholar] [CrossRef] [PubMed]
  146. Vermes, I.; Haanen, C.; Steffens-Nakken, H.; Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 1995, 184, 39–51. [Google Scholar] [CrossRef] [Green Version]
  147. Glander, H.J.; Schaller, J. Binding of annexin V to plasma membranes of human spermatozoa: A rapid assay for detection of membrane changes after cryostorage. Mol. Hum. Reprod. 1999, 5, 109–115. [Google Scholar] [CrossRef]
  148. Makker, K.; Agarwal, A.; Sharma, R.K. Magnetic activated cell sorting (MACS): Utility in assisted reproduction. Indian J. Exp. Biol. 2008, 46, 491–497. [Google Scholar]
  149. Aziz, N.; Buchan, I.; Taylor, C.; Kingsland, C.R.; Lewis-Jones, I. The sperm deformity index: A reliable predictor of the outcome of oocyte fertilization in vitro. Fertil. Steril. 1996, 66, 1000–1008. [Google Scholar] [CrossRef]
  150. Aziz, N.; Said, T.; Paasch, U.; Agarwal, A. The relationship between human sperm apoptosis, morphology and the sperm deformity index. Hum. Reprod. 2007, 22, 1413–1419. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  151. Said, T.; Agarwal, A.; Grunewald, S.; Rasch, M.; Baumann, T.; Kriegel, C.; Li, L.; Glander, H.J.; Thomas, A.J., Jr.; Paasch, U. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: An in vitro model. Biol. Reprod. 2006, 74, 530–537. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  152. Grunewald, S.; Reinhardt, M.; Blumenauer, V.; Said, T.M.; Agarwal, A.; Abu Hmeidan, F.; Glander, H.J.; Paasch, U. Increased sperm chromatin decondensation in selected nonapoptotic spermatozoa of patients with male infertility. Fertil. Steril. 2009, 92, 572–577. [Google Scholar] [CrossRef] [PubMed]
  153. Dirican, E.K.; Ozgün, O.D.; Akarsu, S.; Akin, K.O.; Ercan, O.; Uğurlu, M.; Camsari, C.; Kanyilmaz, O.; Kaya, A.; Unsal, A. Clinical outcome of magnetic activated cell sorting of non-apoptotic spermatozoa before density gradient centrifugation for assisted reproduction. J. Assist. Reprod. Genet. 2008, 25, 375–381. [Google Scholar] [CrossRef] [Green Version]
  154. Said, T.M.; Grunewald, S.; Paasch, U.; Rasch, M.; Agarwal, A.; Glander, H.J. Effects of magnetic-activated cell sorting on sperm motility and cryosurvival rates. Fertil. Steril. 2005, 83, 1442–1446. [Google Scholar] [CrossRef]
  155. Vaughan, D.A.; Sakkas, D. Sperm selection methods in the 21st century. Biol. Reprod. 2019, 101, 1076–1082. [Google Scholar] [CrossRef]
  156. Sackmann, E.K.; Fulton, A.L.; Beebe, D.J. The present and future role of microfluidics in biomedical research. Nature 2014, 507, 181–189. [Google Scholar] [CrossRef]
  157. Cho, B.S.; Schuster, T.G.; Zhu, X.; Chang, D.; Smith, G.D.; Takayama, S. Passively driven integrated microfluidic system for separation of motile sperm. Anal. Chem. 2003, 75, 1671–1675. [Google Scholar] [CrossRef]
  158. Schuster, T.G.; Cho, B.; Keller, L.M.; Takayama, S.; Smith, G.D. Isolation of motile spermatozoa from semen samples using microfluidics. Reprod. Biomed. Online 2003, 7, 75–81. [Google Scholar] [CrossRef]
  159. Ainsworth, C.; Nixon, B.; Aitken, R.J. Development of a novel electrophoretic system for the isolation of human spermatozoa. Hum. Reprod. 2005, 20, 2261–2270. [Google Scholar] [CrossRef] [Green Version]
  160. Shirota, K.; Yotsumoto, F.; Itoh, H.; Obama, H.; Hidaka, N.; Nakajima, K.; Miyamoto, S. Separation efficiency of a microfluidic sperm sorter to minimize sperm DNA damage. Fertil. Steril. 2016, 105, 315–321. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  161. Ohta, A.T.; Garcia, M.; Valley, J.K.; Banie, L.; Hsu, H.Y.; Jamshidi, A.; Neale, S.L.; Lue, T.; Wu, M.C. Motile and non-motile sperm diagnostic manipulation using optoelectronic tweezers. Lab Chip 2010, 10, 3213–3217. [Google Scholar] [CrossRef]
  162. Zhang, X.; Khimji, I.; Gurkan, U.A.; Safaee, H.; Catalano, P.N.; Keles, H.O.; Kayaalp, E.; Demirci, U. Lensless imaging for simultaneous microfluidic sperm monitoring and sorting. Lab Chip 2011, 11, 2535–2540. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  163. Ma, R.; Xie, L.; Han, C.; Su, K.; Qiu, T.; Wang, L.; Huang, G.; Xing, W.; Qiao, J.; Wang, J.; et al. In vitro fertilization on a single-oocyte positioning system integrated with motile sperm selection and early embryo development. Anal. Chem. 2011, 83, 2964–2970. [Google Scholar] [CrossRef] [PubMed]
  164. Xie, L.; Ma, R.; Han, C.; Su, K.; Zhang, Q.; Qiu, T.; Wang, L.; Huang, G.; Qiao, J.; Wang, J.; et al. Integration of sperm motility and chemotaxis screening with a microchannel-based device. Clin. Chem. 2010, 56, 1270–1278. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  165. Ko, Y.-J.; Maeng, J.-H.; Lee, B.-C.; Lee, S.; Hwang, S.Y.; Ahn, Y. Separation of progressive motile sperm from mouse semen using on-chip chemotaxis. Anal. Sci. 2012, 28, 27–32. [Google Scholar] [CrossRef] [Green Version]
  166. Eamer, L.; Vollmer, M.; Nosrati, R.; San Gabriel, M.C.; Zeidan, K.; Zini, A.; Sinton, D. Turning the corner in fertility: High DNA integrity of boundary-following sperm. Lab Chip 2016, 16, 2418–2422. [Google Scholar] [CrossRef]
  167. Ko, Y.J.; Maeng, J.H.; Hwang, S.Y.; Ahn, Y. Design, Fabrication, and Testing of a Microfluidic Device for Thermotaxis and Chemotaxis Assays of Sperm. SLAS Technol. 2018, 23, 507–515. [Google Scholar] [CrossRef]
  168. Matsuura, K.; Uozumi, T.; Furuichi, T.; Sugimoto, I.; Kodama, M.; Funahashi, H. A microfluidic device to reduce treatment time of intracytoplasmic sperm injection. Fertil. Steril. 2013, 99, 400–407. [Google Scholar] [CrossRef]
  169. Samuel, R.; Badamjav, O.; Murphy, K.E.; Patel, D.P.; Son, J.; Gale, B.K.; Carrell, D.T.; Hotaling, J.M. Microfluidics: The future of microdissection TESE? Syst. Biol. Reprod. Med. 2016, 62, 161–170. [Google Scholar] [CrossRef] [Green Version]
  170. Parrella, A.; Keating, D.; Rosenwaks, Z.; Palermo, G.D. A microfluidic device for selecting the most progressively motile spermatozoa yields a higher rate of euploid embryos. Fertil. Steril. 2018, 110, e342. [Google Scholar] [CrossRef] [Green Version]
  171. Tsai, V.F.; Chang, H.-C.; Hsieh, J.-T.; Wo, A.M. Application of microfluidic technologies to the quantification and manipulation of sperm. Urol. Sci. 2016, 27, 56–59. [Google Scholar] [CrossRef] [Green Version]
  172. Nosrati, R.; Vollmer, M.; Eamer, L.; San Gabriel, M.C.; Zeidan, K.; Zini, A.; Sinton, D. Rapid selection of sperm with high DNA integrity. Lab Chip 2014, 14, 1142–1150. [Google Scholar] [CrossRef] [PubMed]
  173. Clark, S.G.; Haubert, K.; Beebe, D.J.; Ferguson, C.E.; Wheeler, M.B. Reduction of polyspermic penetration using biomimetic microfluidic technology during in vitro fertilization. Lab Chip 2005, 5, 1229–1232. [Google Scholar] [CrossRef]
  174. Suh, R.S.; Zhu, X.; Phadke, N.; Ohl, D.A.; Takayama, S.; Smith, G.D. IVF within microfluidic channels requires lower total numbers and lower concentrations of sperm. Hum. Reprod. 2006, 21, 477–483. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  175. Jeon, H.; Cremers, C.; Le, D.; Abell, J.; Han, J. Multi-dimensional-double-spiral (MDDS) inertial microfluidic platform for sperm isolation directly from the raw semen sample. Sci. Rep. 2022, 12, 4212. [Google Scholar] [CrossRef] [PubMed]
  176. Aitken, R.J.; Hanson, A.R.; Kuczera, L. Electrophoretic sperm isolation: Optimization of electrophoresis conditions and impact on oxidative stress. Hum. Reprod. 2011, 26, 1955–1964. [Google Scholar] [CrossRef] [Green Version]
  177. Fleming, S.D.; Ilad, R.S.; Griffin, A.M.; Wu, Y.; Ong, K.J.; Smith, H.C.; Aitken, R.J. Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: Comparison with density gradient centrifugation. Hum. Reprod. 2008, 23, 2646–2651. [Google Scholar] [CrossRef] [Green Version]
  178. Lassalle, B.; Testart, J. Human zona pellucida recognition associated with removal of sialic acid from human sperm surface. J. Reprod. Fertil. 1994, 101, 703–711. [Google Scholar] [CrossRef] [Green Version]
  179. Shapouri, F.; Mahendran, T.; Govindarajan, M.; Xie, P.; Kocur, O.; Palermo, G.D.; Bakos, H.W.; Ahlström, A.; Caisander, G.; Xu, B.; et al. A comparison between the Felix™ electrophoretic system of sperm isolation and conventional density gradient centrifugation: A multicentre analysis. J. Assist. Reprod. Genet. 2023, 40, 83–95. [Google Scholar] [CrossRef]
Sensors 23 06978 g001
Figure 1. Most common non−thermal plasma configurations: (a) dielectric barrier discharge (DBD) plasma and (b) plasma jet.
Figure 1. Most common non−thermal plasma configurations: (a) dielectric barrier discharge (DBD) plasma and (b) plasma jet.
Sensors 23 06978 g001
Sensors 23 06978 g002
Figure 2. Magnetic-activated cell sorting (MACS) overview. (a) The separation columns are surrounded by a magnetic field and loaded with semen mixed with annexin V labeled microbeads. The magnetic field will retain apoptotic spermatozoa tagged to the microbeads while non-apoptotic spermatozoa will flow through the column. (b) Once non-apoptotic spermatozoa are collected, the column is flushed, and apoptotic spermatozoa are washed out.
Figure 2. Magnetic-activated cell sorting (MACS) overview. (a) The separation columns are surrounded by a magnetic field and loaded with semen mixed with annexin V labeled microbeads. The magnetic field will retain apoptotic spermatozoa tagged to the microbeads while non-apoptotic spermatozoa will flow through the column. (b) Once non-apoptotic spermatozoa are collected, the column is flushed, and apoptotic spermatozoa are washed out.
Sensors 23 06978 g002
Sensors 23 06978 g003
Figure 3. Sperm microelectrophoresis overview. Heathy and mature sperm are charged negatively and under electric current will migrate to the anode (positive electrode) and will be separated from immature or damaged sperm. At the same time, spermatozoa are small enough to pass through a polycarbonate membrane and will be separated from other cell types present in semen.
Figure 3. Sperm microelectrophoresis overview. Heathy and mature sperm are charged negatively and under electric current will migrate to the anode (positive electrode) and will be separated from immature or damaged sperm. At the same time, spermatozoa are small enough to pass through a polycarbonate membrane and will be separated from other cell types present in semen.
Sensors 23 06978 g003
Table 1. A summary of antibiotic resistance profiles of bacteria recovered from human and animal semen.
Table 1. A summary of antibiotic resistance profiles of bacteria recovered from human and animal semen.
SubjectsSampleOutcomesRef.
Men with or without urogenital inflammationFresh semenOut of the isolated Gram-positive microorganisms, 33.7% were resistant to tetracycline and 46.9% were resistant to sulfamethoxazole-trimethoprim. In the case of Gram-negative microorganisms, 51.5% were resistant to ampicillin, 43.5% to piperacillin, 25.0% to amoxicillin-clavulanic acid, 23.4% to ciprofloxacin, and 20.3% to levofloxacin.[48]
Fertile and infertile menFresh semen78.9% Escherichia coli isolates were resistant to tetracycline, 73.6% to levofloxacin, 57.8% to tobramycin or chloramphenicol. A total of 66.6% of Klebsiella pneumoniae isolates were resistant to gentamicin, tobramycin, tetracycline, and cefotaxime. A total of 80.0% Staphylococcus epidermidis isolates were resistant to gentamycin and 60% to streptomycin. [49]
Normozoospermic menFresh semenA total of 35.0% Enterococcus faecalis isolates were resistant to ampicillin. A total of 75.0% Staphylococcus haemolyticus isolates were resistant to tobramycin or tetracycline. A total of 70.0% Staphylococcus hominis and 32.0% Staphylococcus capitis isolates were resistant to tobramycin. A total of 68.0% Staphylococcus epidermidis were resistant to tetracycline.[13]
Men with primary infertilityFresh semenHigh resistance of Gram-positive bacteria to rifampicin (48.0% isolates), clindamycin (40.0% isolates), and vancomycin (44.0% isolates). High resistance of Gram-negative bacteria to ceftazidime (20.0% isolates) and cefuroxime (40.0% isolates). [50]
Infertile men with urogenital inflammationFresh semenHigh Staphylococcus aureus resistance to ceftazidime and ceftriaxone (53.9% isolates), fluoroquinolone (69.3% isolates), cefotaxime (42.3% isolates), and oxacixicillin (61.5% isolates). High Escherichia coli resistance to ceftazidime (55.6% isolates), lindamycin and cefotaxime (66.7% isolates), and oxacixicillin (77.8% isolates). High Klebsiella spp. resistance to ceftriaxone (60.0% isolates) and oxacixicillin (90.0% isolates). High Pseudomonas aeruginosa resistance to ceftazidime, ofloxacine, and oxacixicillin (83.3% isolates), gentamycin, vancomycin, and cefotaxime (66.7% isolates).[51]
Men undergoing fertility evaluationFresh semenA rising trend for streptococci resistance to erythromycin. A rising trend for staphylococci to penicillin and ampicillin/sulbactam.[52]
Bovine studsFresh, cooled, and packed semen A total of 23.6% out of the isolated microorganisms presented with resistance to gentamicin, while 92.7% were resistant to streptomycin. Resistance to lincomycin/spectinomycin was observed for more than 23.6% of all isolates. A total of 22% of all isolates were resistant to all tested antibiotics.[53]
Yearling bullsFresh semenAll Serratia liquefaciens and Serratia quinivorans isolates were resistant to cefazolin. All Cronobacter sakazakii, Escherichia fergusonii, and Shigella boydii were resistant to tetracycline. 2 Shigella and 1 Salmonella isolates were resistant to cefazolin, amikacin, and gentamicin.[54]
Breeding ramsFresh semenOne Staphylococcus equorum and one Staphylococcus vitulinus isolate were resistant to ciprofloxacin.[15]
Breeding ramsFresh and diluted semenA total of 53% of all isolates were resistant to penicillin, 47% to erythromycin, 33% to oxytetracycline, 20% to ampicillin, 15% to streptomycin, 15% to co-trimoxazole, 13% to polymixin B, and 4% to spectinomycin.[55]
Duroc boarsDiluted semenAll Enterococcus hirae, Staphylococcus aureus, as well as 50% of Staphylococcus simulans and Staphylococcus chromogenes isolates, were resistant to tigecycline. All Acinetobacter iwoffi, Pseudomonas aeruginosa, and Pseudomonas putida isolates (100%) were resistant to ciprofloxacin.[32]
Breeding boarsFresh semenA total of 56.5% out of the isolated microorganisms presented with resistance to gentamicin and penicillin, while 58.7% were resistant to neomycin. Resistance to ceftiofur and lincomycin was observed for more than 47.8% of all isolates. A total of 50.0% of all isolates were resistant to ampicillin.[56]
Breeding boarsFresh and diluted semenEscherichia coli isolates showed 50% resistance to amikacin and 70% resistance to gentamicin; Staphylococcus epidermidis was 12.5% resistant to amikacin and 50% to gentamicin; Serratia marcescens showed a resistance of 66.6% to amikacin and 50% to gentamicin; Proteus spp. isolates were 25% resistant to amikacin and 50% to gentamicin; Streptococcus spp. isolates were 50% resistant to gentamicin; Staphylococcus aureus isolates were 100% resistant to gentamicin.[57]
Big 6 turkeysFresh semenAll Staphylococcus lentus isolates were resistant to chloramphenicol, linezolid, and tigecycline. Enterococcus faecium isolates showed resistance against imipenem, while ertapenem was shown to be ineffective against Escherichia coli and Vagococcus fluvialis isolates.[16]
Breeding turkeysFresh and stored semenAll Enterococcus faecalis, Bacillus subtilis and corynebacteriaisolates were resistant to ampicillin-cloxacillin, cefuroxime, amoxicillin, and ceftriaxone. All Escherichia coli isolates were resistant to co-trimoxazole, ofloxacin, and nalidixic acid.[58]
Lohmann Brown and ROSS 308 roostersFresh semenCitrobacter braakii (75% isolates), Enterococcus faecalis (25% isolates), Escherichia coli (46% isolates), and Staphylococcus epidermidis (20% isolates) were resistant to ampicillin. Resistance to tetracycline was observed in Staphylococcus epidermidis isolates (60%), while 36% Escherichia coli isolates were resistant to chloramphenicol. Multiresistance patterns against several antibiotics were recorded, particularly in the cases of Citrobacter braakii, Enterococcus faecalis, Escherichia coli, and Staphylococcus epidermidis.[17]
Broilers, layers, and fattening turkeysFresh semenHigh resistance rates were identified in Enterococcus faecalis and Enterococcus faecium for lincomycin (72–99% isolates) and tetracycline (67–82% isolates). More than half of Enterococcus isolates were resistant to gentamicin (54–72% isolates), erythromycin (44–61% isolates), and tylosin (44–56% isolates). At total of 89 out of 145 Enterococcus isolates were resistant to three or more antimicrobial classes. [59]
Indian red jungle fowlCryopreserved semenEscherichia coli showed high resistance towards streptomycin. Staphylococcus spp. isolates were resistant to kanamycin. Bacillus spp. isolates were resistant to neomycin and streptomycin[60]
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Ďuračka, M.; Benko, F.; Chňapek, M.; Tvrdá, E. Strategies for Bacterial Eradication from Human and Animal Semen Samples: Current Options and Future Alternatives. Sensors 2023, 23, 6978. https://doi.org/10.3390/s23156978

AMA Style

Ďuračka M, Benko F, Chňapek M, Tvrdá E. Strategies for Bacterial Eradication from Human and Animal Semen Samples: Current Options and Future Alternatives. Sensors. 2023; 23(15):6978. https://doi.org/10.3390/s23156978

Chicago/Turabian Style

Ďuračka, Michal, Filip Benko, Milan Chňapek, and Eva Tvrdá. 2023. "Strategies for Bacterial Eradication from Human and Animal Semen Samples: Current Options and Future Alternatives" Sensors 23, no. 15: 6978. https://doi.org/10.3390/s23156978

APA Style

Ďuračka, M., Benko, F., Chňapek, M., & Tvrdá, E. (2023). Strategies for Bacterial Eradication from Human and Animal Semen Samples: Current Options and Future Alternatives. Sensors, 23(15), 6978. https://doi.org/10.3390/s23156978

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop