Amikacin/Penicillin Saw Enhanced Antimicrobial Activity in Semen Extender

By Marie Rosenthal, MS

The addition of amikacin and penicillin to a commercial semen extender enhanced antimicrobial activity against commensal bacteria without affecting the sperm quality or fertility, according to several studies performed by Camilo HernándezAvilés, DVM, of Texas A&M University.

It is normal for the skin of the penis of breeding stallions to harbor nonpathogenic bacteria, and most ejaculates contain some commensal bacteria. However, sometimes ejaculates are contaminated with potentially pathogenic bacteria, usually Klebsiella pneumoniae or Pseudomonas aeruginosa. So, antibiotics are frequently added to inhibit bacterial growth, explained Dr. Hernández-Avilés, a PhD student in the Department of Large Animal Clinical Services at the College of Veterinary Medicine.

A variety of commercially available semen extenders and several antibiotics are used for this purpose, but Dr. Hernández-Avilés and his group wanted to know whether 2 more narrow-spectrum antibiotics—amikacin and penicillin—would be as effective as other antibiotic types, such as synthetic β-lactams. A 2018 study from his group found that amikacin and penicillin had better antimicrobial activity than new generation antibiotics—synthetic β-lactams or carbapenems—when added to a milkbased semen extender. In that study, they found that both commensal bacteria and certain levels of potentially pathogenic bacteria were completely eliminated from semen when the extender contained a conventional dose of both amikacin and penicillin. Yet, when the ejaculates were contaminated with high levels of P. aeruginosa¸ the addition of amikacin and penicillin did not offer any advantage over the other antibiotics tested. From that study, they concluded that none of the antibiotic additions affected the sperm quality, and the use of amikacin and penicillin in a milk-based extender provided “better control of commensal bacterial populations when semen was cooled.”

A culture taken from raw semen of a stallion with bacterial urethritis. The bacteria obtained included a mixed growth of both Escherichia coli and Klebsiella pneumoniae.

Source: Dr. Hernández-Avilés

A culture plate obtained from cooled stored semen diluted with INRA-96 extender with added amikacin and penicillin and looked at (3,000 ug/mL–3,000 IU/mL).

Source: Dr. Hernández-Avilés

In their current study, they wanted to determine the best doses to inhibit bacterial growth without affecting the sperm motion characteristics and plasma membrane intactness of semen diluted with a very popular casein-based extender in North America and Europe, INRA-96. This semen extender is formulated with certain antibiotics that are added at lower concentrations than in other commercially available semen extenders. “The INRA-96 extender is a very effective medium to preserve semen quality during cooled storage; however, we have some field reports of heavy growth of pathogenic bacteria when this extender is used for cooled storage of semen,” he explained. So they wanted to know if adding amikacin and penicillin to this casein-based extender could offer better control of bacterial growth without affecting sperm quality.

They collected semen from multiple stallions and then diluted it with INRA-96 extender to 3 treatment groups: 1. amikacin/penicillin G, 2. ticarcillin clavulanate (both at standard doses), and 3. no additional antibiotics and served as a control.

They checked the sperm quality after 30 minutes and then again after 24 hours of cooled storage. There was less growth in the INRA-96 plus amikacin/penicillin group than the other 2 groups, he said.

“We found that after 30 minutes of exposure or 24 hours of cooled storage, amikacin/penicillin added to the INRA-96 extender significantly reduced the number of commensal bacteria as compared with ticarcillin clavulanate or the extender with no added antibiotic,” he explained, and the sperm quality did not appear to be affected.

The amikacin they were using was expensive, so they wanted to switch to a less expensive product by another company, and repeated the experiment to ensure that the second amikacin was also effective, substituting the less expensive amikacin with the more expensive product in a fourth group.

“We concluded that the amikacin products yielded similar values for semen quality and bactericidal effectiveness versus commensal bacteria, but again, we're still having some commensal bacterial growth,” Dr. Hernández-Avilés said.

“We decided to do a dose-titration experiment in which we wanted to find the most effective dose of amikacin/penicillin to completely eliminate commensal bacteria growth, without affecting sperm quality,” he said.

They collected semen from multiple stallions, which was diluted with the INRA-96 extender and looked at 5 different amikacin/penicillin dosing group: • a standard dose of 1,000 µg/mL of amikacin and 1,000 IU/mL of penicillin, • or 2, 3, 4, 5 times this standard dose.

They found that with doses of 3,000 µg/mL of amikacin and 3,000 IU/mL of penicillin, “there was a significant improvement on the complete elimination of commensal bacteria as compared with the standard dose or a 2-fold dose,” he said. This improvement was seen at 30 minutes, as well as 24 hours after cold storage. The sperm quality was not affected in any treatment group.

“Still you might wonder what could happen with the fertility of semen that has been processed with these types of antibiotics,” he said, so they did 1 more study, in which cooled semen was used to impregnate 11 broodmares.

The semen was donated by 1 stallion and was divided into 2 treatment groups: 1. the INRA-96 extender without additional antibiotics, 2. the INRA-96 extender with 5,000 µg/mL of amikacin and 5,000 IU/mL of penicillin added.

A pregnancy diagnosis was conducted 14 days after ovulation by ultrasonography. They could not detect any significant differences between both treatment groups in terms of pregnancy rate.

“So, based on the results from this study and previous studies from our laboratory, a standard dose of amikacin/penicillin was not completely effective for controlling commensal bacteria or potentially pathogenic bacteria,” he said, but a higher dose was more effective.

Because the studies used commensal rather than pathogenic bacteria, the studies are now being done looking at K. pneumoniae and P. aeruginosa contaminants.

Doses this high might not be preferred routinely if there are no pathogenic bacteria, because of concerns about resistance, Dr. Hernández-Avilés admitted. However, these 2 organisms can cause bacterial endometritis in mares, and resistance for amikacin is relatively rare in veterinary medicine.

In their recent study published in the journal Theriogenology, they included data from 2 stallions whose semen was heavily contaminated with K. pneumoniae or P. aeruginosa. They found that adding amikacin and penicillin to the INRA-96 extender helped to control bacterial growth in the ejaculates from these stallions, without compromising sperm quality.

“Amikacin/penicilin—even at high doses—can be considered safe for sperm and fertility and are more effective than new-generation antibiotics when we are faced with commensal bacteria or some potentially pathogenic bacteria,” he said.

Dr. Hernández-Avilés presented some of this work at the annual convention of the American Association of Equine Practitioners

For more information:

Hernández-Avilés C, Love CC, Serafini R, et al. Inclusion of supplemental antibiotics (amikacin-penicillin) in a commercial extender for stallion semen: Effects on sperm quality, bacterial growth, and fertility following cooled storage. Theriogenology. 2020;158:209–217. https://www.sciencedirect.com/science/article/abs/pii/S0093691X20305112?via%3Dihub

Hernández-Avilés C, Love CC, Serafini R, et al. The effects of antibiotic type and extender storage method on sperm quality and antibacterial effectiveness in fresh and cooled-stored stallion semen. Theriogenology. 2018; 122:23–29. https://www.sciencedirect.com/science/article/pii/S0093691X18303625