Equine Semen Extenders: Effect on Bacterial Growth and Sperm Viability

Preliminary results of a collaborative effort between researchers at Oklahoma State University and Texas A&M University were recently presented at the American Association of Veterinary Laboratory Diagnosticians in San Diego, Calif. These researchers compared antibiotic properties of common equine semen extenders with four different strains of bacteria, including Taylorella equigenitalis, the causative organism for contagious equine metritis (CEM).

Unique to this study were the comparisons of how these extenders and bacteria affect semen quality and longevity.

Equine semen that is packaged and cool-shipped for artificial insemination (AI) is almost always extended with a skim milk- or egg yolk-based semen extender. There are multiple types of these extenders available on the market, but results on their benefits and detriments are not definitive.

In equids, they are utilized for their ability to maintain plasma membrane integrity while protecting spermatozoa from cold shock, detrimental seminal plasma proteins, and toxic byproducts produced by the sperm cells. Most extenders used today also contain antibiotics to decrease bacterial growth in semen; however, additional antimicrobial supplementation of extender is also accepted.

Unfortunately, proper evaluation of extenders is difficult because they contain compounded ingredients and might be proprietary. Despite this variance, they are essential for the preservation of equine semen.

Both natural and artificial insemination can lead to uterine infections, which are a significant cause of reduced fertility in the mare. Four organisms commonly isolated from equine bacterial endometritis cases are Streptococcus equi subsp. zooepidemicus, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumonia. In addition, the recent outbreak of CEM poses further questions about the risk of T. equigenitalis associated with cool-shipped semen and the potential for post-breeding uterine infections. It was the recent outbreak and the on-going quarantines and investigation that lead to this study. Semen being exported from the United States is required to be extended and shipped in extenders capable of killing the CEMO.

The effect of bacterial growth in extenders is not well-documented. For that reason the study investigated the potential for bacterial growth in cool-shipped stallion semen using specific extenders with known concentrations of specific bacterial pathogens, and the impact of these specific extenders and bacteria on semen quality and longevity.

Experimental Model

For this experiment, the researchers used INRA 96, a widely used skim milk-based manufactured extender containing the antibiotics penicillin, gentamicin, and amphotericin B. It was used as supplied by the manufacturer and also used with several concentrations of an added antibiotic (Timentin--ticarcillin disodium and clavulanate potassium).

Two other semen extenders, VMDZ and E-Z Mixin "BF", were also used. VMDZ is an egg yolk-based extender manufactured with amikacin and penicillin G potassium, and E-Z Mixin “BF” or “Basic Formula” is a skim milk-based extender produced with no antibiotics added, used as the antibiotic-free extender control group.

This collaborative research effort resulted in several interesting findings. INRA 96, as supplied by the manufacturer, was found to allow more bacterial growth over 72 hours than it did with Timentin added. However, VMDZ extender consistently had minimal, to no, bacterial growth.

While INRA 96 was found to maintain sperm motility and viability better than VMDZ, T. equigenitalis grew in INRA 96 (no Timentin added) about half of the time at 24 hours of refrigeration, which is equivalent to an ove-night semen shipment.

VMDZ semen extender had superior antimicrobial properties compared to the other extenders used in this study, yet did not perform quite as well in maintaining sperm motility and viability over the 72 hour test period. The complete results of this study are in the process of publication.

This poster was entitled "Bacterial Growth and Sperm Viability in Equine Semen Extenders Inoculated with Specific Bacteria, Including Taylorella equigenitalis (the causative bacteria for contagious equine metritis)."

Lead presenter on this poster was Bettina Olivieri, DVM, theriogenology intern, Department of Clinical Sciences, College of Veterinary Health Sciences, Oklahoma State University, Tina.Olivieri@okstate.edu. Others involved in this research were Brenda Love, Grant Rezabek, and Cathy Lamm of the Oklahoma Animal Disease Diagnostic Laboratory; Sheila R. Teague and Dickson Varner, Department of Large Animal Clinical Sciences, College of Veterinary Medicine, Texas A&M University; and Reed Holyoak, Department of Clinical Sciences, Oklahoma State University.

About the Author

Kimberly S. Brown

Kimberly S. Brown was the Publisher/Editor of The Horse: Your Guide To Equine Health Care from June 2008 to March 2010, and she served in various positions at Blood-Horse Publications since 1980.

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