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¹Atatürk University Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Erzurum, Turkey
²Elbistan Vocational School, Kahramanmaras İstiklal University, Kahramanmaras, TR
³Department of Medical Microbiology, Faculty of Medicine, Ataturk University, Erzurum, TR
⁴Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, TR
⁵Food and Livestock Application and Research Center, Ataturk University, Erzurum, TR
⁶Atatürk University Faculty of Veterinary Medicine, Department of Biochemistry, Erzurum, Turkey
⁷Ataturk University, Faculty of Science, Department of Molecular Biology and Genetics, Erzurum, Turkey

*Corresponding author: ¹Ali Dogan Omur, Ataturk University Faculty of Veterinary Medicine, Department of Reproduction and Artificial Insemination, Erzurum, Turkey
²Demet Celebi, Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, TR

Abstract

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Microbiota is a cluster of physiological and pathogenic microorganisms found in many tissues and organs of living things, including such as bacteria, viruses, fungi. Microbiota has important roles on many system functions such as immune system, urinary system, digestive system. However, these microorganisms can cause obstruction in the genital tract, epididymitis and orchitis, and thus may lead to infertility. In addition, pathogenic and apathogenic microorganisms such as bacteria, viruses and fungi have negative effects on spermatology. Decreased sperm motility, disruption of membrane integrity, and acrosome damage are the most common of these negativities. Approximately 15% of male infertility in the world is associated with infection in the genital tract. The most common microorganisms found in the male genital tract and semen are Enterobacter, Lactobacillus and Staphylococci. In addition, microorganisms in semen cause infections in the female genital tract, birth of offspring with anomalies and embryonic deaths. In this study, the current literature on the microorganisms found in the male genital tract and semen in different species has been compiled.
Keywords: Infertility; Microbiota; Semen
Abbreviations: GU: Genitourinary, STD: Sexually Transmitted Diseases, ART: Assisted Reproductive Technologies, CFU: Colony Forming Units, LAB: Lactic Acid Bacteria, IPV: Infectious Pustular Vulvovaginitis, IBR: Infectious Bovine Rhinotracheitis, IPB: Infectious Pustular Balanopostitis, FMD: Foot and Mouth Disease, BT: Bluetongue, BVD: Bovine Viral Diarrhea, BL: Bovine Leukemia, EF: Ephemeral Fever, LSD: Lumpy Skin Disease

Introduction

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The terms abiogenesis and biogenesis were explained by Thomas Henry Huxley (1825 - 1895). He proposed that the term abiogenesis be used to mean the spontaneous process of formation, and the term biogenesis to be used to refer to the process of life arising from similar life. Microorganisms were discovered in the late 17th century by Robert Hooke and Antoni van Leeuwenhoek [1].
Microbiota; while it contributes to the development of the immune system, has important roles on skin, urinary system, respiratory system, digestion and absorption of nutrients, production of vitamins, development and functions of the gastrointestinal immune system, the living body also provides a rich nutrient environment for the survival of microorganisms. Although there have been studies explaining the role of microbiota in the organism in recent years, it is important to create optimum health conditions of the body by understanding the molecular mechanisms in more detail with the point reached today.
Previous studies have defined that microbiota in the organism is related to homeostasis and health [2,3] including male and female genital tracts [4]. Microbiomes play an important role in disease and some etiologies. The skin, intestines, oral cavity, vagina, and urethra are among the sites that host microbial communities. The composition of the microbiomes can alter its effect on metabolism [5-7].
Microbiota in Semen
Sexually transmitted diseases (STDs) can affect individual morbidity, mortality and fertility [8]. Semen is not sterile and may contain microorganisms even after processing for Assisted reproductive technologies (ART) [9]. Infections in the genitourinary (GU) canal account for up to 15% of the causes of male infertility [10]. In the sequencing studies performed in the genitourinary system of men and women, genes of pathogenic factors were found, and it was stated that these factors have negative effects on the health of the reproductive system [11]. Studies have showed that a large number of infectious bacteria, viral, fungal and protozoan species can enter the normal genital-urinary tract through sexual transmission, intracanalicular spread of infected urine [12,13]. The bacterial microbiome is referred to as the extracellular microenvironmental component [14]. Also, bacteria can have a direct negative impact on spermatozoa physiology, reducing viability or motility and decrease mitochondrial activity [15-18] One of the most important causes affecting spermatogenesis is infections that directly affect the testicles [19,20]. These infections prevent the continuity of spermatogenesis. It can affect spermatozoa as well as cause defects in the formed spermatozoa [21]. Some infections, inflammation in the tissues, enlargement of the genital tract, epididymitis and orchitis may be related to male infertility [22]. Microorganism load in semen increases the number of macrophages and polymorphonuclear granulocytes, forming the primary line of defense. This defense mechanism leads to the production of reactive oxygen species (ROS) in the semen, and thus, with a large increase in the number of dead spermatozoa, ROS production also increases and the semen begin to lose functions [23-25].
Genital Mycoplasma (Mycoplasma genitalium and Mycoplasma hominis) colonizes the genitourinary tract of both males and females [26]. M.genitalium caused sperm agglutination and decreased motility in sperm in vitro. It is thought that this situation may also affect fertilization [27].
Various microorganisms cause problems such as balanitis, postitis, seminal vesiculitis, prostatitis, urethral inflammations, testicular degenerations, orchitis, epididymitis, ampullitis, resulting in male infertility [28]. Microorganisms in semen can be viruses, bacteria, chlamydia, rickettsia or fungi and are generally classified as pathogenic, potentially pathogenic, and nonpathogenic [29].
Semen is an important vector for the spread of viral infections. Some viruses can be found in semen cells (leukocytes, macrophages, sperm) and free semen [30]. Hepatitis B, C, HIV, Papillomavirus, Herpesvirus, Cytomegalovirus can spread through semen in humans [27]. In addition, in humans, Trichomonas vaginalis is a protozoan and causes infection around the urethra, especially since it prefers squamous cells [31].
Various microorganisms, including viruses, can be found in semen due to infections. These agents can be transmitted to susceptible animals by artificial and natural insemination. This causes the infection to be transmitted to animals inseminated with semen and even to encounter various consequences affecting the embryo [32]. The bacterial load in fresh semen is between 104 and 106 colony forming units (CFU) /mL [33]. Bacterial load in semen often leads to loss of sperm motility and sperm agglution and sperm plasma membrane damage, resulting in infertility and economic damage [34].
In the figure below (Figure 1), the ratio information about bacterial microorganisms in (A) normal semen and (B) case samples is given.

Probiotics are defined as live microorganisms that confer a health benefit on the host [35]. The most commonly used probiotics are lactobacilli and bifidobacteria, which produce lactic acid as the primary metabolite of sugar metabolism. Lactobacilli and bifidobacteria strains have also been reported to produce antioxidants [36]. Lactobacillus, which contains about 180 species, is not known to have any negative effects on the living thing. Lactic acid bacteria (LAB) such as Lactobacillus, Lactococcus, Pediococcus, Streptococcus and Enterococcus are in this class [37,38]. Studies have shown that lactic acid bacteria (LAB) in the genus Lactobacillus spp. prevent pathogenic microorganisms that may occur, and that they can do this by consuming resources, stimulating the host immune system, decreasing the pH, and producing acetic and lactic acid with hydrogen peroxide [39,40]. Depending on the species, environmental factors can also contaminate sperm in semen examination and analysis in the laboratory [41].
The presence of bacteria in pig semen has been demonstrated by studies [42].
Brucellosis, Chlamydophilosis, Leptospirosis and Enterobacteriaceae are among the subjects studied in pig semen [43,44]. A study has shown that Lactobacillus can be found in pig semen and its prolonged incubation will have a negative effect on spermatozoa [45].
Artificial insemination is one of the most frequently used methods in dogs [46]. Semen is not a sterile secretion as it contains physiological microbial flora from the dog's urogenital tract [47; Osborne and Lees, 1995). Fractions present in canine ejaculate stimulate bacterial growth. Escherichia coli, Staphylococcus aureus, Klebsiella spp., Mycoplasma canis, Pseudomonas aeruginosa and Streptococcus spp. bacteria are more common in the first fraction of dog’s ejeculate [47,48]. Bacterial growth of less than 105 colony forming units (CFU)/mL in ejaculate from dogs is considered physiological, while high values are a cause of pathological risk [49,50].
Various exogenous and endogenous factors affect semen quality in rams. Bacteria found in ram ejaculates can originate from different sources such as wool, skin, urine, feces. However, contaminated feed, water, and poor hygiene conditions in the production area may increase bacterial contamination of semen [51]. Aeromonas veronii, Bacillus subtilis, Enterobacter bugandens, Escherichia coli, Escherichia hermannii, Klebsiella variicol, Lactobacillus curvatus, Mannheimia haemolytica, Providencia rettgeri, Pseudomonas lutea, Staphylococcus delphini, Staphylococcus capitis, Staphylococcus chromogenes, Staphylococcus sciura, Staphylococcus simulans and Staphylococcus vitulinus are bacteria found in the ram semen [52]. In a study performed before mating in rams, swap samples were taken from the glans penis and bacteria such as
Staphylococcus aureus, Streptococcus pyogenes, Proteus mirabilis and Brucella abortus were isolated [53]. In horses, Staphylococcus spp, Micrococcus spp, Pseudomonas spp were isolated in semen [54]. Taylorella equigenitalis is located in the distal part of the genital tract in horses and can remain there for a long time [55].
In poultry, pathogenic microorganisms that can potentially contaminate semen are transmitted through the cloaca [56]. Salmonella spp. Campylobacter spp., Staphylococcus spp., Coliform spp., Streptococci spp. and Bacillus spp. are among the bacteria found in the ejaculate of poultry [57]. Similarly in a study conducted in turkeys, only Enterobacter spp. was isolated from semen [58].
In fish semen, bacteria of the genus Pseudomonas spp are expressed as part of the microbiota of the semen [59, 60]. In a study, Argyrosomus regius, Flavobacterium spp., Aeromonas spp. and Corynebacterium spp., Pseudomonas spp. and Vibrio sp. are isolated [61].
Microbiological control of ejaculates and spermatozoon count in straws aim to eliminate the prevalence of bacteriospermia from 7% to 99% of ejaculates [62-66]. While bacteria such as Bacterioidetes, Actinobacteria, Proteobacteria, Firmicutes, Fusobacteria or Cyanobacteria are present in the semen of bulls, sometimes opportunistic pathogenic bacteria such as Staphylococcus, Streptococcus, Mycoplasma, Pseudomonas, Corynebacterium or Bacillus can also be found [67,68]. Among the viruses isolated from bovine semen; Infectious Bovine Rhinotracheitis (IBR), Infectious Pustular Vulvovaginitis (IPV) Irifexiosis Pustular Balanopostilis (IPB), Foot and Mouth Disease (FMD), Bluetongue (BT), Bovine Viral Diarrhea (BVD), Bovine Leukemia (BL), Ephemeral Fever (EF), and Lumpy Skin Disease (LSD) viruses can be counted [69-76].
It was determined that Chlamydia, Campylobacter, Chlamydophila were found in preputial swabs of goats [77]. In a study conducted in goats, it was determined that the bacterial load increased in goats over 5 years old, the rainy season increased the number of bacteria, and Jamunapari goats had a higher bacterial load [78].
In a study of Australian saltwater crocodile, an unidentified Pseudomonas species Citrobacter braakii, Enterobacter agglomerans, Klebsiella ornitholytica, Klebsiella pneumonia, Proteus Aeromonas veronii bio sobria, Bacillus cereus, Chryseosepticia bacterium avium, Providenceosepticia cocciantobacterium, Providetobacterium Enterosepticia cocciantuca mirabilis, Pseudomonas aeruginosa, Salmonella arizonae were detected in penile shaft, sulcus and semen [79]. In koalas, Corynebacterium species were found both in the foreskin and semen [80]. On the other hand, Staphylococcus spp. and Bacillus spp. were reported to be among the main isolated micro-organisms in camel [81]. In a study conducted in Pecari tajacu, Corynebacterium spp, Staphylococcus spp, Bacillus sp, Rhodococcus spp, Dermabacter spp, Microbcterium spp species were found in semen and foreskin [82].
Another work conducted in rabbits, Proteobacteria, Firmicutes, Fusobacteria and Bacteroidetes were found in the sperm microbiota. It has been determined that host genetics influence the bacterial community composition in the sperm microbiota. In addition, in their discriminant analysis, they stated that Lysinibacillus and Flavobacterium are biomarkers for fertility [83].

Conflict of interest: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding: The authors received no financial support for the research, authorship, and/or publication of this article.
ORCID ID: Ali Doğan ÖMÜR https://orcidorg/0000-0002-2976-4368
ORCID ID: Serkan Ali AKARSU https://orcidorg/0000-0003-4450-6540
ORCID ID: Büşra AYYILDIZ https://orcidorg/0000-0001-5116-1400
ORCID ID: Demet ÇELEBİ https://orcidorg/0000-0002-2355-0561
ORCID ID: Mehmet Akif AYDIN https://orcidorg/0000-0002-6238-018X

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