Antimicrobial resistance of the microbiota in the reproductive tract of horses in conjunction with assisted reproduction

Antimicrobial resistance is a global challenge exacerbated by antibiotic misuse and inadequate infection control. This study explores the effects of antibiotics in semen extenders on the vaginal bacteria of the mare and investigates the separation of spermatozoa from bacteria in seminal plasma as an alternative to adding antibiotics to semen extenders. In addition, it examines the diversity of the seminal microbiota in healthy stallions across different countries. Antimicrobial resistance was influenced by breeding status and age of the mare, as well as environmental factors such as housing and location, and exposure to antibiotics in semen extenders. There were geographic differences in the diversity of the seminal microbiota, with implications for tailored antibiotic use. While phylum level composition of seminal microbiota remains consistent, genus level variations may stem from husbandry or individual differences. Bacterial load could be reduced by Single Layer Centrifugation with a low density colloid, without compromising sperm quality. This method has potential for circumventing the need for antibiotics in semen extenders, although further research is needed to confirm sperm fertilizing capacity

Practical Method for Freezing Buck Semen

Although several protocols for cryopreserving buck semen are described in the literature, they differ widely in factors such as season and method of semen collection, extender and sperm concentration. Therefore, choosing a protocol that is suitable for a particular on-farm situation can be problematic. In the present study, semen was collected by artificial vagina from seven bucks on a farm located approximately 90 minutes’ drive away from the laboratory, about 6 weeks before the start of the goat breeding season. The semen was immediately extended in warm semen extender containing soy lecithin and was placed in an insulated box with a cold pack for up to 4 h, during semen collection from the remaining bucks and subsequent transport to the laboratory. Following centrifugation at 4 °C and resuspension in the soy lecithin extender to a sperm concentration of 800 × 106 spermatozoa/mL, 0.25 mL plastic straws were filled and frozen in racks 4 cm above the surface of liquid nitrogen. This simple protocol resulted in an acceptable post-thaw quality for all seven bucks, with a mean post-thaw motility of 55 ± 21% and mean fragmented chromatin of 3.27 ± 1.39%. Normal sperm morphology was >90% in all ejaculates. The semen was sent to a gamete bank for long-term storage

Antimicrobial Resistance in Vaginal Bacteria in Inseminated Mares

Antimicrobials are added to semen extenders to inhibit the growth of bacteria that are transferred to the semen during collection. However, this non-therapeutic use of antimicrobials could contribute to the development of antimicrobial resistance. The objective of this study was to determine changes in the antibiotic susceptibility of vaginal microbiota after artificial insemination. Swabs were taken from the vagina of 26 mares immediately before artificial insemination and again 3 days later. Bacteria isolated from the vagina at both time points were subjected to antibiotic susceptibility testing and whole-genome sequencing. In total, 32 bacterial species were identified. There were increases in the resistance of Escherichia coli to trimethoprim (p = 0.0006), chloramphenicol and (p = 0.012) tetracycline (p = 0.03) between day 0 and day 3. However, there was no significant effect of exposure to antibiotics in semen extenders with respect to the resistance of Staphylococcus simulans and Streptococcus equisimilis (p > 0.05). Whole-genome sequencing indicated that most phenotypic resistance was associated with genes for resistance. These results indicate that the resistance patterns of vaginal bacteria may be affected by exposure to antibiotics; therefore, it would be prudent to minimize, or preferably, avoid using antibiotics in semen extenders

Antibiotic resistance patterns in cervical microbes of gilts and sows

Extenders for boar semen contain antibiotics, which may induce antimicrobial resistance (AMR) in inseminated females. The objective was to investigate AMR of bacteria isolated from the cervix of sows and gilts in standing heat, representing females previously exposed to antibiotics in the semen extender and non-exposed females, respectively. Cervical swabs were taken from 30 multiparous sows and 30 gilts prior to their first insemination. After culturing on agar plates, bacterial isolates were identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry and antimicrobial minimum inhibitory concentrations (MIC) were determined. Differences in antibiotic resistance between sows and gilts were analyzed by Chi-squared or Fisher’s exact test. Bacteria isolated were mostly Staphylococcus spp., Streptococcus spp. and Corynebacterium spp. Higher MICs were observed for isolates from sows than from gilts. Most (>80%) Corynebacterium spp. were resistant to clindamycin; small numbers (<20%) were resistant to gentamicin, penicillin, vancomycin, ciprofloxacin and rifampicin, with no differences between gilts and sows. Corynebacterium from gilts were more often resistant to tetracycline than those from sows (25% vs. 4.17%; p = 0.04). In conclusion, bacteria from the porcine cervix showed low resistance to most antibiotics except for clindamycin, but antibacterial resistance may increase with increasing parity

Antimicrobial resistance in equine reproduction

Bacteria develop resistance to antibiotics following low-level “background” exposure to antimicrobial agents as well as from exposure at therapeutic levels during treatment for bacterial infections. In this review, we look specifically at antimicrobial resistance (AMR) in the equine reproductive tract and its possible origin, focusing particularly on antibiotics in semen extenders used in preparing semen doses for artificial insemination. Our review of the literature indicated that AMR in the equine uterus and vagina were reported worldwide in the last 20 years, in locations as diverse as Europe, India, and the United States. Bacteria colonizing the mucosa of the reproductive tract are transferred to semen during collection; further contamination of the semen may occur during processing, despite strict attention to hygiene at critical control points. These bacteria compete with spermatozoa for nutrients in the semen extender, producing metabolic byproducts and toxins that have a detrimental effect on sperm quality. Potential pathogens such as Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa may occasionally cause fertility issues in inseminated mares. Antibiotics are added during semen processing, according to legislation, to impede the growth of these microorganisms but may have a detrimental effect on sperm quality, depending on the antimicrobial agent and concentration used. However, this addition of antibiotics is counter to current recommendations on the prudent use of antibiotics, which recommend that antibiotics should be used only for therapeutic purposes and after establishing bacterial sensitivity. There is some evidence of resistance among bacteria found in semen samples. Potential alternatives to the addition of antibiotics are considered, especially physical removal separation of spermatozoa from bacteria. Suggestions for further research with colloid centrifugation are provided

Practical Method for Freezing Buck Semen

Although several protocols for cryopreserving buck semen are described in the literature, they differ widely in factors such as season and method of semen collection, extender and sperm concentration. Therefore, choosing a protocol that is suitable for a particular on-farm situation can be problematic. In the present study, semen was collected by artificial vagina from seven bucks on a farm located approximately 90 minutes&rsquo; drive away from the laboratory, about 6 weeks before the start of the goat breeding season. The semen was immediately extended in warm semen extender containing soy lecithin and was placed in an insulated box with a cold pack for up to 4 h, during semen collection from the remaining bucks and subsequent transport to the laboratory. Following centrifugation at 4 &deg;C and resuspension in the soy lecithin extender to a sperm concentration of 800 &times; 106 spermatozoa/mL, 0.25 mL plastic straws were filled and frozen in racks 4 cm above the surface of liquid nitrogen. This simple protocol resulted in an acceptable post-thaw quality for all seven bucks, with a mean post-thaw motility of 55 &plusmn; 21% and mean fragmented chromatin of 3.27 &plusmn; 1.39%. Normal sperm morphology was &gt;90% in all ejaculates. The semen was sent to a gamete bank for long-term storage

Alternatives to Antibiotics in Semen Extenders Used in Artificial Insemination

Antimicrobial resistance is a serious global threat requiring a widespread response. Both veterinarians and medical doctors should restrict antibiotic usage to therapeutic use only, after determining the sensitivity of the causal organism. However, the addition of antibiotics to semen extenders for animal artificial insemination represents a hidden, non-therapeutic use of antimicrobial substances. Artificial insemination for livestock breeding is a huge global enterprise with hundreds of million sperm doses prepared annually. However, reporting of antimicrobial resistance in semen is increasing. This review discusses the consequences of bacteria in semen samples, as well as the effect of antimicrobial substances in semen extenders on bacteria in the environment and even on personnel. Alternatives to antibiotics have been reported in the scientific literature and are reviewed here. The most promising of these, removal of the majority of bacteria by colloid centrifugation, is considered in detail, especially results from an artificial insemination study in pigs. In conclusion, colloid centrifugation is a practical method of physically removing bacteria from semen, which does not induce antibiotic resistance. Sperm quality in stored semen samples may be improved at the same time

Antibiotic Resistance Patterns in Cervical Microbes of Gilts and Sows

Extenders for boar semen contain antibiotics, which may induce antimicrobial resistance (AMR) in inseminated females. The objective was to investigate AMR of bacteria isolated from the cervix of sows and gilts in standing heat, representing females previously exposed to antibiotics in the semen extender and non-exposed females, respectively. Cervical swabs were taken from 30 multiparous sows and 30 gilts prior to their first insemination. After culturing on agar plates, bacterial isolates were identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry and antimicrobial minimum inhibitory concentrations (MIC) were determined. Differences in antibiotic resistance between sows and gilts were analyzed by Chi-squared or Fisher&rsquo;s exact test. Bacteria isolated were mostly Staphylococcus spp., Streptococcus spp. and Corynebacterium spp. Higher MICs were observed for isolates from sows than from gilts. Most (&gt;80%) Corynebacterium spp. were resistant to clindamycin; small numbers (&lt;20%) were resistant to gentamicin, penicillin, vancomycin, ciprofloxacin and rifampicin, with no differences between gilts and sows. Corynebacterium from gilts were more often resistant to tetracycline than those from sows (25% vs. 4.17%; p = 0.04). In conclusion, bacteria from the porcine cervix showed low resistance to most antibiotics except for clindamycin, but antibacterial resistance may increase with increasing parity

Vaginal Bacteria in Mares and the Occurrence of Antimicrobial Resistance

Antibiotics are added to semen extenders in insemination doses but their effect on the vaginal microbiota of the inseminated female is unknown. The objectives of this study were to define the equine vaginal microbiota and its antimicrobial resistance, and to determine whether it changes after exposure to antibiotics in semen extenders. Vaginal swabs were taken prior to sham-insemination (day 0), and again on days 3, 7, and 14 after insemination. Isolated bacteria were identified by MALDI-TOF and tested for antimicrobial susceptibility by microdilution. The bacteria isolated from the vagina differed according to reproductive status (brood mare or maiden mare), location (north or middle of Sweden), and the stage of the estrous cycle. Five bacterial species were frequently isolated from mares in both locations: Escherichia coli, Staphylococcus capitis, Streptococcus equisimilis, Streptococcus thoraltensis, and Streptococcus zooepidemicus. Overall, vaginal bacteria isolated from inseminated mares showed higher antibiotic resistance than from non-inseminated mares, suggesting a possible link between exposure to antibiotics in the semen extender and the appearance of antimicrobial resistance. The whole-genome sequencing of E. coli isolates from inseminated mares revealed some genes which are known to confer antimicrobial resistance; however, some instances of resistance in these isolates were not characteristic of induced AMR