Prepare Your Mare For Mating  - What Do Uterine Cultures Really Mean?

By Juan C. Samper DVM, PhD Dipl. ACT and Tracy A. Plough DVM

Every spring, horse breeders with mares that were bred but did not become pregnant the previous year are confronted with the decision of whether or not to try again. Owners of such mares often think that they have done all they can and have exhausted all possibilities, which most times involve a culture, cytology, and some uterine treatment.

Endometritis, which is the inflammation (swelling and irritation) of the uterus, is the main reason for mares not becoming pregnant. This inflammation can be acute or it can be chronic; in other words, this inflammation could be something that is occurring for the first time or it could be a problem that has been going on for a long time. 

The most critical component in preventing this problem is the mare herself. A mare whose reproductive tract is normal in both form and function will be able to quickly evacuate excess fluids from the uterus after breeding and after foaling. However, this evacuation can be hindered by anatomical abnormalities such as the vulvar angle, pendulous uterus, incompetent cervix, or degeneration of the uterus, making it unable to contract or to drain.

Endometritis is the primary reason for mares not becoming pregnant. Main Photo: Shutterstock/Kwadrat

Endometritis can also be influenced by the presence of one or several bacteria. These bacteria can cause different types of problems and have developed ways to evade the immune response of the mare and the antibiotics we use. Consequently, endometritis can result in a variety of clinical signs as well as ultrasonographic and laboratory findings. For example, bacteria such as E coli adhere tightly to the inner lining of the uterus, preventing their physical removal, while others such as streptococcus stimulate production of an inflammatory exudate with the consequent production of large amounts of pus. Others such as Pseudomonas aeruginosa and some yeast and fungi secrete a biofilm, which is an adhesive, cellophane type film that supports growth and maintenance of bacterial micro-colonies. Biofilms provide inherent resistance to antibiotics resulting in persistent, chronic infections even after prolonged antibiotic treatment. Some bacteria or fungi form focal plaques that are not identified by routine swab culture techniques, while others don’t produce intrauterine fluid, the hallmark of endometritis. 

The conformation of a mare’s reproductive tract greatly contributes to whether or not breeding her will be successful. Anatomical abnormalities can lead to the inability to properly flush excess fluids, leading to endometritis. Photo: Robin Duncan Photography

The uterine response to a pathogen also contributes to the establishment of long-term or chronic infections. 

Because of the many factors affecting fertility, veterinarians need to tailor their diagnostics specifically for each individual. Recent information indicates that ultrasonographic and cellular findings from mares with endometritis differ between the types of bacteria present and that traditional uterine swabs have lower chances of identifying bacteria compared to small volume uterine flushings or endometrial biopsies.

Related: Breeding Your Mare

Classical treatments for endometritis include uterine irrigation, administration of drugs to contract the uterus, and intra-uterine infusion of antibiotics. These treatments are not effective in all cases of endometritis. Proper use of products such as mucolytics and steroids can increase pregnancy rates in chronically infected mares. In addition, intra-uterine infusion with some agents can break and penetrate the biofilm and are successful in clearing chronic gram negative and yeast endometritis that did not respond to routine therapy.

Veterinarians need to examine problem mares on more than one occasion, since often clinical signs are expressed over time and we need to identify a pattern to determine if the mare is normal or not. Ultrasonography and several modalities of culturing and cellular evaluation are used for the purpose of diagnosing and then treating the mare appropriately. 

Ultrasonography

Accumulations of uterine fluid prior to breeding or shortly after are indicators of reduced pregnancy rates and that the mare has problems. However, intra-uterine fluid is not always associated with the presence of bacteria in the uterus. For example, mares with uterine bacteria such as E. coli, Staphylococcus aureus, Pseudomonas spp., or others considered non-pathogens had intrauterine fluid in less than 40 percent of ultrasonographic examinations. On the other hand intra-uterine fluid was seen more frequently in half of the mares (45 to 55 percent) when β-hemolytic Streptococcus, Klebsiella, Enterobacter cloacae, or yeast were isolated. 

An ultrasound showing uterine edema or inflammation. Photo courtesy of Dr. Juan Samper

Other ultrasonographic abnormalities associated with decreased pregnancy rates include abnormal edema patterns such as excessive edema pre- or post-mating, an edema pattern that does not extend throughout the uterine wall, or the presence of short, thick lines within the uterine wall, signifying either air or thick pus.

Modalities of Uterine Cultures and Cytology

Traditional uterine culture and cytology are the most common techniques for diagnosing endometritis, by detecting the presence of bacteria and/or inflammatory cells (neutrophils). If a uterine pathogen is isolated and the mare has more than 2 neutrophils per 400 x field (a positive uterine cytology), she can reliably be diagnosed as having endometritis. However, mares may also yield positive uterine cytologies with negative uterine cultures, or mares can have positive cultures with no inflammation present.

Until recently the dogma was that we needed to have both positive culture and positive cytology in order to consider the mare infected. However, recent research has shown unequivocally that positive culture OR positive cytology are associated with endometritis and decreased pregnancy rates. The type of organism determines the level of inflammation that the mare has.

A cytology showing white blood or inflammatory cells. Photo courtesy of Dr. Juan Samper

The type of bacteria and the length of time that it has been in the uterus will determine how easy it is to culture. Bacteria that associated with uterine fluid are more likely to have inflammatory cells (neutrophils) on cytology, while bacteria not associated with uterine fluid tend to be negative for neutrophils on cytology. So the type of diagnostic procedure becomes a critical part of determining what the mare has. Four options are available: culture of biopsy, swab culture, low volume flush culture, and cytology.

Of these techniques, low volume flush and biopsy cultures are about three times more sensitive than traditional swabs or cytologies alone in diagnosing uterine infections. This becomes very relevant when a mare continually cultures negative (false negative) on a traditional swab and does not get pregnant on multiple breeding cycles or attempts with or without fluid accumulation or discharges. 

In a more recent study performed on a large group of mares, it was concluded that inflammation based on neutrophil presence or presence of bacteria together or independently had a negative effect on pregnancy rates of mares. So it is critical that the attending veterinarian takes into account the clinical signs in order to interpret laboratory information, since the correlation between cytology and culture results varies between microorganisms recovered. Furthermore it is important to realize that: 1) a positive culture, neutrophilic cytology, or abnormal flush alone would indicate a mare as having endometritis, and 2) the type of microorganism recovered does not determine if the bacteria is significant or not if the sample was taken with proper technique. 

Related: Breeding with Cooled Semen

In the previous section I described the multiple reasons why mares become infected and why they are difficult to diagnose. Without proper diagnosis it becomes very difficult to properly treat a truly infected mare.

So what should we do if we find an infected mare? The most important point in establishing a successful therapy is to have an adequate diagnosis of the problem. Therapy of problem or infected mares is based on three major principles: correcting the defects in uterine defense, neutralizing virulent bacteria, and controlling post-breeding inflammation. This is accomplished by surgically correcting anatomical defects, improving physical drainage after insemination, reducing the length of or modulating the inflammatory response to insemination, and inhibiting bacterial growth. In some cases, the uterus is infused with antibiotics post mating.

New Treatment Strategies

Not all infections respond to uterine irrigation and antibiotic treatment. Treatment failure may be due to continual contamination of the uterus because of anatomical abnormalities in the perineal area, breakdown of antibiotics in the uterus, or biofilm production by the bacteria. Determination of mucus production and treatment with mucolytic agents such as DMSO and N-acetylcysteine added to the treatment fluids has become an integral part of treatments in an attempt to clear exudate, mucus, or biofilm.

Antibiotic failure in chronic endometritis may be due to biofilm produced by some gram negative bacteria, yeast, and fungi. Bacterial biofilms consist of a mixed community of different bacterial species, surrounded by an extracellular matrix, that co-exist together forming a group of “persister cells” that are essentially invulnerable to antibiotics and that neither grow or die but maintain a persistent infection. Common bacteria known to produce biofilms include but are not limited to Pseudomonas spp, Staphylococcus epidermis, E. coli, Enterobacter and a number of yeast. Work in other species and in the mare has shown that buffered chelating agents (Tris-EDTA, Tricide®) may potentiate the actions of antimicrobials, dissolve exudate, and break up biofilm.

Related: Equine Embryo Transfer

Every procedure done in the uterus (breeding infusion, lavage, etc.) causes a certain degree of inflammation. It is also known that certain mare categories such as older or fat mares tend to have exaggerated inflammatory responses. Modulation of the inflammatory response with dexamethasone or prednisolone has become a key factor in mare infertility treatment. However, candidates for steroid use should be chosen carefully as misuse in mares with bacterial endometritis may exacerbate the infection.

Summary

Not all uterine infectious agents produce the classic signs of uterine inflammation such as fluid production and white blood cell migration and may therefore require diagnostic methods other than culture swab for identification. As pathogens induce different uterine responses and have developed different methods for evading the uterine defense mechanism of the mare, treatment with mucolytics or buffered chelating agents can improve treatment success. Steroids can also be used to modulate or help the mare deal with the uterine response to insemination. In order to implement the appropriate treatment it is critical to have a proper diagnosis. Treating all problem mares with everything will only make the problem worse.

Dr. Juan Samper, MSc, PhD, Diplomat ACT, operated JCS Veterinary Reproductive Services in Langley, BC, from 1993-2017. He has consulted with breeders and veterinarians in over 25 countries. He served as the Associate Dean Clinical Affairs at Ross University School of Veterinary Medicine from 2014 to 2017, and is presently the Associate Dean of Students and the University of Florida College of Veterinary Medicine. 

Main Photo: Shutterstock/Kwadrat