Equine Breeding Industry Updates

Some mares with infertility problems have an infection in the uterus that can simmer along without recognition. This hide-and-seek game with dormant bacteria can be difficult to target with appropriate therapy. Many strains of bacteria hide within biofilms they produce and antibiotics can’t reach them. (A biofilm is formed by a group of bacteria that stick together and are embedded within a self-produced extracellular matrix of “slime” that protects them from surrounding host defenses.)

Credit: Thinkstock There have been many advances in equine reproduction, and some “old” fertility treatments are regaining popularity.

Lisa Metcalf, MS, DVM, DACT (Diplomate of the American College of Theriogenologists), is a Research Assistant Professor at Oregon Health Science and University and owner of Honahlee PC, an equine reproduction facility in Sherwood, Oregon. She stressed, “Sometimes mares are treated unnecessarily by vets and non-vets with an indiscriminate use of post-breeding uterine treatments that include infusion with low-dose antibiotics.”

She stressed that every study done to date has not shown any advantage to indiscriminate post-breeding infusion with antibiotics. “There are cases where it is appropriate, but most of the time it is an unnecessary procedure,” she said. “Pregnancy rates are not increased with this treatment, which should be directed by bacterial culture and antibiotic sensitivity.”

Uterine infections can come from inoculation of bacteria from the vagina, which is not sterile. A normal mare has barriers to this inoculation and can clear inflammatory debris should it occur. But infusing the uterus is an invasive enough procedure that bacteria can be introduced unnecessarily. This not only prolongs the inflammatory reaction in the uterus following breeding, but using low-dose antibiotics also adds to the potential for development of resistant bacteria. “Use of antibiotics should be implemented only for a reason and following bacterial culture and antibiotic sensitivity testing,” urged Metcalf.

Metcalf noted that in normal mares a uterine swab sample taken for cytology (cell evaluation) during estrus would be negative for the presence of neutrophils, a type of white blood cell that targets bacterial invasion. Yet in the case of a mare infected with biofilm-producing bacteria, the same sample that is negative for neutrophils on cytology might grow a pure colony of E. coli. “Possibly when taking the sample, the biofilm is scraped away to reach the colony of bacteria beneath it. The biofilm protected the underlying bacteria from a host immune response,” remarked Metcalf.

The current problem with overuse of antibiotics is based on the concern that it stimulates bacterial resistance to our present armamentarium of anti-microbial medications. No new antimicrobial medications are being developed in the near future. This worldwide health issue has sparked greater interest in treatments that rely on non-antibiotic therapies.

Previous non-antibiotic treatments that have been used are now being reinstated because of their effectiveness. Many of these treatments are used without antibiotic therapy, while others are used in addition to implementation of antibiotics based on bacterial culture and sensitivity results. Mares with compromised uterine defense mechanisms, delayed uterine clearance, or post-breeding endometritis are good candidates for some of these new and “new again” techniques:

  • Metcalf finds that uterine infusion with platelet rich plasma (PRP) has a significant effect on modulating the horse’s immune system and is especially useful for mares with delayed uterine clearance of fluid. When mares with post-mating delayed uterine clearance are bred, this inflammatory reaction is caused by the presence of semen. PRP inhibits interleukin 1B and other inflammatory cytokines while also boosting anti-inflammatory cytokines. She believes it also has an effect on nitric oxide to improve uterine perfusion (blood circulation)–this is important for healing. In addition, PRP is bactericidal for many organisms.
  • With inflammation, the uterus secretes excess mucus. This not only interferes with antibiotic penetration but also may create a barrier to sperm transport to the oviduct. For this reason, treatment with an anti-mucolytic, like n-acetylcysteine (NAC) can modulate the amount of mucus by decreasing its viscosity. NAC has also been shown to have anti-microbial and anti-oxidative properties.
  • DMSO (dimethyl sulfoxide) is another mucolytic agent that breaks down mucus. In addition, it is a potent anti-inflammatory medication when infused into the uterus as a dilute saline solution. Metcalf reports that it may work to interrupt bacterial biofilms that protect the bacteria against the host’s immune defenses. Mares receiving a dilute DMSO/saline solution post-breeding had higher pregnancy rates than mares receiving only saline. Uterine biopsy findings were improved in the treated mares.
  • bActivate is a new product that is intended to provide a growth medium to “activate” dormant Streptococcal bacteria that reside deep within the uterine lining. This product is administered directly into the uterus. By turning a dormant infection into an active one, it is more likely that appropriate antibiotics can address the infection in addition to the host immune system recognizing a need to respond. There are reports of its effectiveness in enabling diagnosis and subsequent uterine treatment but it has not yet been tested and compared against control mares receiving a placebo.
  • EDTA-tris (Tricide) is a buffered chelator that, in vitro (in the lab), appears to poke holes in bacteria and biofilms, dissolves exudates, and enables penetration of the biofilm by antibiotics. One group of bacteria that has demonstrated in vitro susceptibility to EDTA-tris is E. coli. It is thought that direct contact with this chelating solution removes calcium and magnesium from the bacterial outer cell wall thereby increasing permeability and decreasing stability of the cell wall. This leads to bacterial cell death.
  • Cationic steroid antimicrobial (CSA) treatment is reported to have antibacterial, antiviral, anti-fungal, and anti-biofilm properties. One product containing CSA is called Ceragyn although in vitro  testing reports that it does not disrupt bacteria although it is yet to be tested in the horse.
  • The development of long-acting intramuscular carbetocin (Duratocin) is helpful for mares that are desensitized to oxytocin, which is used to stimulate uterine contractions to help clear the uterus of fluid post-breeding. These products are not for sale in the USA and the veterinarian must have a special license to bring them in from out of the country.
  • Prostaglandin F2a (Estrumate or Lutalyse) also causes uterine contraction but Metcalf advises that it should not be used after ovulation because it may adversely affect the corpus luteum necessary to maintain a conceptus.
  • For anti-inflammatory effects on the uterus, corticosteroids may be useful but it has been shown in some studies that dexamethasone lowers luteinizing hormone (LH), which triggers ovulation and the development of the corpus luteum. It can safely be given before ovulation but not after, says Metcalf. By lowering LH, dexamethasone may elicit an anovulatory cycle, i.e., no egg ovulates off the ovary. Studies have demonstrated that a different corticosteroid–prednisolone–can be given both pre-breeding and post-breeding with minimal effects on LH.
  • In vitro studies have caused clinicians to reconsider the bactericidal effects of hydrogen peroxide, reports Metcalf.
  • Infusion of kerosene has also been used for chronic infertility and might function by breaking up protective biofilms produced by certain bacterial and fungal pathogens.

Coordinating Timing of Estrus Cycles

One effective method of inducing a mare to come into heat early in the spring is to put her under lights in the stall. To stimulate hormonal changes that favor development of an ovulatory follicle, a mare must be exposed to 16 hours of light followed by 8 hours of darkness. Other studies have demonstrated that 1-2 hours of light following the onset of darkness interrupts melatonin secretion as a method of shortening the time until the first ovulation of the season.

The Equilume Light Mask ( is a hood containing a half cup over one eye that shines a blue light at specific hours–controlled by an automatic timer–to serve the same effect as bringing a mare into a stall under lights, but without having to resort to using a stall. The mare can stay in pasture day and night. The Mask contains a light sensor that is activated when environmental light levels drop at dusk, and the blue light then stays on for a seven-hour period. The biggest drawback is the price since the non-replaceable battery only lasts a single season so it is necessary to replace the entire hood.

Low dose deslorelin, injectable progesterone/estrogen, or 10-14 days of Regumate (altrenogest) can also stimulate cyclicity during transition in early spring. If either of the latter steroid products is used, they might be followed by a prostaglandin analog injection eight days later to bring the mare into heat. This technique can enable scheduling of semen collection for insemination at the optimal time.

Another method used to hasten the time to seasonal estrus cycles is with hormonal treatment using a long-acting estradiol cypionate (ECP) combined with a dopamine antagonist (domperidone or sulpiride) given 24 hours later. In one study, it was shown that treated mares ovulate within 32 days of this combined, one-time treatment.

Investigation has examined another means of coordinating timing of estrous cycles in mares: A controlled implant device (CIDR) is inserted into the vagina where it releases progesterone +/- estradiol over 10-14 days. Unfortunately, there is some concern about the implant causing an intense vaginitis.

Another significant finding has shown that shipped semen is not as fertile at 24 hours as when it is inseminated immediately following collection. Mare owners using shipped semen need to be aware of this.

In Vitro Fertilization

“In vitro fertilization (IVF) is another way to preserve genetics,” said Metcalf. Historically, this has been a difficult method to achieve fertilization in the lab in horses. It was thought that sperm is not adequately activated to penetrate the thick zona pellucida that surrounds an egg.

Currently, an in vitro fertilization process exists with a new twist: (ICSI) intracytoplasmic sperm injection whereby a veterinarian aspirates immature oocytes from a mare’s ovarian follicles via ultrasound-guided trans-vaginal aspiration (TVA) techniques. Although this process has been used for years, what is novel about it is that no longer does the mare have to be shipped to the reproduction facility. The oocyte can be acquired at the mare’s home base and only the oocytes shipped overnight to the lab. Once at the lab, the oocytes are incubated to maturation and a single sperm is injected into each oocyte. The fertilized oocyte is cultured in the lab to mature into an embryo, which occurs within 6-9 days. The embryonic blastocyst is then shipped back to the veterinarian for non-surgical embryo transfer into the uterus of a recipient mare. Or, the embryo can be frozen and stored for future use.

Metcalf reported that the ICSI procedure is greatly helpful when sperm numbers are “constrained” due to inadequate sperm production by a sub-fertile stallion or the stallion has died and there is very little sperm available. It is also valuable in mares with uterine or oviductal compromise that are unable to produce a pregnancy even through embryo transfer. This ICSI procedure is performed in very few locations in the USA: Colorado State University Equine Reproduction Lab; Texas A & M University; Equine Medical Services in Columbia, Missouri; InFoal Inc in Hemet, California; and OHSU Equine IVF Lab in Portland, Oregon.

For endangered breeds of horses, those with fertility problems, or for horses that have died and there is a limited quantity of frozen semen, the ICSI procedure might be invaluable for perpetrating a genetic line. Embryos and semen can be frozen indefinitely and then used at the opportune time.






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