Molecular characterization of Staphylococcus species isolates from buck semen and their effect on semen quality

INTRODUCTION

In the present scenario, goat artificial insemination (AI) is gaining importance, but the success of AI in goats is dependent on the efficient semen cryopreservation techniques. Bacterial contamination can compromise the quality of buck semen due to its spermicidal effects. Bacteria are present in every semen ejaculate; most of them being commensal bacteria, but some of them may be harmful pathogens. However, by following proper precautions, ensuring healthy and hygienic maintenance of breeding bucks can minimize the contamination and hence will aid in production of quality semen.^[1,2] Staphylococcus aureus is the most common Gram-positive bacteria and it is also involved in variety of infections.^[3] Various virulence factors include protein A, Panton Valentine Leukocidin, coagulases, certain toxins such as surface and pore forming toxins, superantigens, and entertoxins that aids in establishing successful disease conditions in host.^[4]

Male genital tract is exposed to the environment and use of artificial vagina (AV) and other semen collection tools could act as fomites in spreading the infection. However, use of quality tools with proper hygiene and maintenance of animals in clean environment could minimize the affections of pathogens. Hence, a mechanism is required to screen the bucks for presence of pathogens and its regular screening. In the present study, we have bacteriologically tested the buck semen for the presence of various staphylococcal species that can cause possible affections in the semen quality and in turn be a threat to the female fertility and conception rate. Infections of the male genitourinary tract lead to infertility by affecting spermatogenesis, inflammations of male reproductive tract, obstruction of epididymis, and stress leading to poor quality semen.^[5]

These pathogens are also the important carrier of antimicrobial resistance factors such as Methicillin-resistant S. aureus and semen contaminated with such microbes can compromise the safety of personnel involved in the AI. Moreover, these pathogens or other attributes can be a direct threat to the breeding does inseminated through AI. Hence, the experiment was planned to investigate the presence of Staphylococcus isolates from the semen samples of bucks along with their molecular speciation using multiplex Polymerase Chain Reaction (PCR) and their effect on semen quality.

MATERIAL AND METHODS

RESULTS

Isolation and identification of Staphylococcus from buck semen

The Staphylococcus isolates thus obtained were based on the Gram’s staining, growth on mannitol salt agar and catalase test is tabulated [Table 1 and Figure 1]. In this study, we could isolate 25 (52.08%) Staphylococcus spp. isolates from semen samples.

Table 1: Incidence of Staphylococcosis in buck semen.

Breed	Total no. of semen ejaculates tested	Semen ejaculates positive for Staphylococcus no.(%)
Barbari	16	7 (43.75)
Jamunapari	16	8 (50.00)
Jakhrana	16	10 (62.50)
Total	48	25 (52.08)

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Figure 1:

(a) Staphylococcus aureus with lemon yellow colonies on mannitol salt agar showing fermentation of mannitol with characteristic yellow zone around the colonies of. (b) S. aureus appearing as Gram-positive cocci arranged as characteristic “bunch of grapes.”

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DISCUSSION

This study was primarily designed to corroborate the phenotypical parameters of semen vis-à-vis the presence of Staphylococcus spp. in the semen from selected bucks maintained in healthy environment. Furthermore, the effect of Staphylococcus on semen quality was assessed. Various species of Staphylococcus were detected from breeding buck’s semen by multiplex PCR. About 52% of the semen samples obtained from different breeds of goats were found positive for Staphylococcus species which was confirmed by Gram’s staining as well as growth on mannitol salt agar. In Barbari – 43.75%, Jamunapari – 50%, and Jakhrana – 62.50% were found to harbor various species of Staphylococcus in its semen [Table 1]. A similar pattern was also observed in the earlier study in bucks.^[2]

The presence of Staphylococcus in buck semen has a direct effect on the semen, as we found significant reduction in the progressive motility, acrosomal integrity, and plasma membrane integrity and significant increase in sperm abnormalities [Table 2]. In earlier studies, it is reported that S. aureus can easily invade the reproductive tract affecting its functions^[6,13] and reducing the reproductive potential of spermatozoa.^[14] Most studies conducted in the past also reported that S. aureus and S. epidermidis are the most common Staphylococcus spp. reported in bull semen and the metabolic by-products secreted from these species were reported to have damaging effects on the acrosomal integrity and sperm motility.^[15] Similarly, a study conducted on ram semen ejaculates reported the presence of S. aureus and S. epidermidis and other Gram-negative bacteria belonging to enterobacteriaceae were found in higher proportion of semen samples.^[16] Similar studies were conducted in buffalo, which too reported Staphylococcus species in its semen.^[17] S. aureus affects the sperm function by hampering the metabolic activity of spermatozoa and also reducing its viability. We found that the prevalence of S. aureus strains [Table 1] was 52.08 %, which was similar to the previous reports.^[18] In the present study, we could confirm various species of Staphylococcus including S. aureus, S. chromogenes, S. haemolyticus, S. sciuri, S. simulans, and S. epidermidis in the buck semen [Table 3]. The isolation of these organisms and its effect on sperm morphology and function has been poorly investigated globally. Earlier studies reported that aerobic cocci are present in about 50% of semen samples of male partners having fertility related issues.^[19] Diemer et al. (2003) reported that the most commonly isolated bacteria from male animals with urogenital tract infections or semen contamination are Escherichia coli.^[20] However, Enwurua et al. (2016) reported that Staphylococcus spp. is the most predominant microorganism of urogenital infections.^[21] In the present study, besides S. aureus, we have obtained considerable number of isolates of other species including S. chromogenes, S. hemolyticus, S. simulans, and S. sciuri by molecular detection.

In the present study, there is significant decrease in progressive motility, acrosomal integrity, and plasma membrane integrity in bacterial contaminated semen [Table 2]. In concurrence to the present findings, earlier, Huwe et al. (1998) studied the effect of different pathogenic microorganisms on human sperm motility parameters and found that S. aureus negatively influences sperm motility.^[17,22] In another study conducted by Liu et al. (2018) also reiterated the fact that semen quality is deteriorated while infected with S. aureus.^[18] The declined spermatozoa motility may be due to the inhibition of ATPase pump of spermatozoa.^[23] Earlier researchers have reported that bacterial interaction with spermatozoa hampers the sperm motility,^[24-26] while some researchers have reported that bacteria in semen are responsible for the production of soluble spermicidal factor in the extracellular medium which is responsible for the reduced sperm viability and acrosomal damage.

CONCLUSION

The association of a specific bacteria and its effect in semen quality has been a topic of least importance especially in domestic animals. In this study, we tried to examine the effect of various Staphylococcus species on semen quality parameters. The phenotypical effect such as decrease sperm motility and the bacterial moieties can damage acrosomes and cause DNA damage by producing reactive oxygen species leading to poor conception rates and eventually affecting the financial returns.