Top Equine Reproduction Studies of 2014

Photo: Anne M. Eberhardt/The Horse

To kick-start the educational sessions at the 2014 American Association of Equine Practitioners' (AAEP) Convention, held Dec. 6-10 in Salt Lake City, Utah, three veterinarians presented their favorite surgery, medicine, and reproduction studies from the past year. On the breeding side, researcher and Texas A&M University theriogenology professor Terry Blanchard, DVM, Dipl. ACT, described 12 equine reproduction-related papers sure to impact the industry.

Treating Biofilm Formation in Mares with Uterine Infections

Researchers from Colorado State University evaluated the effects of three different treatments on bacterial biofilm (the extracellular matrix that bacteria produce to resist antimicrobials' effects) production in vitro. In their study they exposed three bacterial isolates (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) cultured from clinical cases to three antibiofilm substances (acetylcysteine [ACE], gallium nitrate, and TRIS-EDTA solution). Their goal was to see if any of these substances could reduce the amount of biofilm produced. While no single treatment consistently prevented biofilm formation in all isolates, they found that all treatments were effective against E. coli but not against K. pneumoniae. These "may reduce biofilm protection of bacteria in chronic uterine infections," Blanchard summarized.

N-Acetylcysteine's Effect on Sperm

A group of researchers conducted a multi-center study to determine what concentration of N-acetylcysteine (NAC, a mucolytic sometimes used to help treat endometritis, an inflammation of the uterus' inner lining) administration in mares just prior to insemination would not affect sperm. In doing so, they mixed NAC solutions in varying concentrations with either chilled or frozen semen. They determined that 0.5-1% NAC did not significantly inhibit the sperm's viability or motility over 24 hours of storage. "If you must breed a mare shortly after treatment with NAC, the authors suggested the use of 0.5-1% NAC to avoid adverse effects on sperm," Blanchard said.

CCFA's Efficacy as Endometritis Treatment

In this experimental study, Colorado State University researchers evaluated ceftiofur crystalline free acid (CCFA) levels in the endometrium after intramuscular administration. They administered the drug to three groups of mares (at 6.6mg/kg body weight) at various intervals and collected blood and endometrial biopsy samples. Upon analyzing these, the researchers determined that CCFA will maintain endometrial levels above the minimum inhibitory concentration (the lowest concentration of drug required to inhibit the growth of a bacterial isolate) for Streptococcus zooepidemicus, the most common cause of infectious endometritis in the mare, for up to 6 days.

Using Acute Phase Proteins to Monitor Placentitis

Researchers from the University of Kentucky hypothesized that certain types of proteins become elevated in mares with placentitis (inflammation of the placenta, an important cause of pregnancy loss). To verify this, they measured concentrations of the proteins serum amyloid A (SAA) and haptoglobin (Hp), as well as white blood cell counts (WBC) and fibrinogen concentrations in mares with experimentally induced placentitis. They determined that SAA and Hp levels increased significantly after induced infection and remained high until the mares aborted. Fibrinogen concentrations and WBC counts, on the other hand, were not useful markers. Blanchard said these results raise the question of whether veterinarians can use SAA or Hp screening to identify at-risk mares earlier in the course of the disease.

Effect of PRP on Post-Mating Endometritis

Persistent post-breeding endometritis is a frustrating cause of reduced fertility in mares. In an effort to provide veterinarians with another treatment option for this condition, researchers evaluated autologous (derived from the horse's own blood) platelet-rich plasma's (PRP) effects on mares' uterine inflammatory response when infused into the uterus after artificial insemination. Researchers have used PRP in orthopedic settings to increase growth factor expression for tissue repair and to modulate the inflammatory response. In this study the team found that, in susceptible mares, uterine fluid, nitric oxide levels, and percentage of neutrophils all decreased after PRP treatment four hours post-breeding. These measurements indicate a reduced inflammatory response, leading Blanchard to conclude that PRP might provide another effective treatment option for this condition.

Comparing Autologous Platelet Concentrate Preparation Techniques

On a related topic, Blanchard described a study in which researchers aimed to compare five autologous platelet concentrate (APC, used to enhance regeneration in tissues that have poor natural healing capabilities) preparation techniques originally developed for humans and now routinely used in horses. They found marked variation among a number of important enrichment factors that could influence APC's activity in biological systems and, thus, clinical outcome. The authors concluded that veterinarians should not rely on the manufacturer's data relating to human patients when selecting an appropriate method for horses. "Many of these techniques … are not nearly as effective (in horses) as they have been reported to be in enhancing platelets and platelet factors in humans,” Blanchard said. "So they may need to be tailored to the horse."

Using Stem Cells to Treat Persistent Mating-Induced Endometritis

Colorado State University researchers set out to determine whether biological treatments, such as autologous conditioned serum (ACS) and mesenchymal stem cells (MSCs), could help modulate the inciting inflammatory response in mares with persistent mating-induced endometritis. They infused either ACS or MSCs into one of two groups of six mares prior to inseminating them with freeze-killed sperm, "which will stimulate quite a uterine reaction," Blanchard noted. The team determined that both treatments decreased the inflammatory response six hours post-insemination. "This is a critical time period because it's about when most studies have found a divergence in the response that occurs in normal mares and mares that seem to be very susceptible to persistent mating-induced endometritis," Blanchard said. The researchers also found that MSCs increased IL-1Ra, which Blanchard said might be very important in helping to control some of the effects of interleukin 1, one of the common pro-inflammatory cytokines produced during post-mating endometritis. Overall, they concluded that stem cells might benefit mares affected by this condition.

Monitoring Supplemental Corpora Lutea Formation in Recipient Mares

A recipient mare used for embryo transfers must be cycling in synchronization with the donor mare in order to be a match for the transfer. When clinics don't have enough embryo recipients whose cycles match with donors, they might administer progestin (trade name Regumate, a synthetic hormone used to suppress or synchronize estrus or maintain pregnancy) to noncycling mares that are to be used as recipients. In the next study Blanchard described, researchers monitored supplementary corpora lutea (SCL, structures that develop in ovarian follicles in response to equine chorionic gonadotropin produced by the endometrial cups from the developing pregnancy) via ultrasound to determine when to cease administering progestin to these noncycling recipient mares. Twenty-five study mares were placed into three groups: progestin treatment for either 70 or 120 days, and controls. The team found that noncycling mares were much slower to develop their first SCL, didn’t form as many SCL, and had lower progesterone production. This was particularly true of mares that had embryos transferred into them while they were in anestrus. The researchers also observed that stopping progestin treatment at 70 days might be premature. Thus, "monitoring SCL formation by ultrasound may be adequate for determining a need for progestin supplementation," Blanchard said.

He said this information is useful even for veterinarians not working with noncycling recipient mares. "Many of the mares we have coming back into our practices from breeding farms are on Regumate," he explained. "One of the first questions your client will ask you is, 'When can I stop the Regumate?' Instead of just coming up with an arbitrary opinion, you can certainly scan for (SCL) when you're evaluating the pregnancy for normalcy."

Quality of Frozen-Thawed Epididymal Sperm vs. Ejaculated Semen

A group of researchers in Brazil compared sperm quality and fertility rates using frozen-thawed epididymal sperm (from the testes) versus ejaculated semen from eight subfertile stallions. They found that the frozen-thawed epididymal sperm's quality was much higher than the ejaculate's, and upon artificial insemination, its pregnancy rates were higher (48%, which "is quite respectable for frozen-thawed semen," said Blanchard) than frozen-thawed ejaculated sperm (23%), as well. The researchers concluded that epididymal sperm were more resistant to freezing than ejaculated sperm. "The reason that I find this so interesting is that they identified this group of subfertile stallions from routine inseminations and low pregnancy rates," Blanchard added. "So it makes me wonder if adverse seminal plasma effects might be more of a problem than we expect."

He cautioned that in Brazil veterinarians use a different freezing extender with different cryoprotectants than do most North American vets, which might have affected the study's outcome.

Cholesterol-Loaded Cyclodextrin's Effect on Cooled Semen

Some practitioners add cholesterol-loaded cyclodextrin (CLC) to the membranes of collected sperm to increase its resistance to freezing. However, cholesterol might also have adverse effects by delaying or inhibiting sperm capacitation and the acrosome reaction required for fertilization to occur. In another study out of Brazil, researchers evaluated CLC's effects on cooled semen (which, said Blanchard, had not previously been studied—only frozen semen) from both a "good" and "bad" cooling stallion and found that it increased pregnancy rates for the poor-cooling stallion. Of the untreated semen, only 44% resulted in a pregnancy, whereas 76% of the CLC-treated semen resulted in a pregnancy. "They did, in fact, demonstrate that the induced acrosome reaction was delayed by 1-2 hours, but it did not adversely affect fertility," said Blanchard. Thus, this simple treatment might provide another method to improve poor-cooling stallions' fertility rates in shipped-semen breeding programs.

Bacteria Transmission from Stallions to Mares Post-Breeding

A group from the University of Kentucky looked at the presence of bacteria on stallions' external genitalia and whether that bacteria were recovered from mares post-breeding with live cover. They used 15 stallions and 206 mares from two Central Kentucky farms. They determined that 22% of the stallions were positive for potentially pathogenic bacteria, yet only 29% of the mares bred to those positive stallions had positive uterine cultures the following day. The researchers also looked for potential correlations between whether a mare cultured positive after being bred to a negative or a positive stallion and found no significant relationship between these factors. They concluded that breeding to stallions with positive cultures did not increase the incidence of positive post-breeding uterine cultures in mares. "This is probably going to be of interest to those of you who are responsible for monitoring stallions in breeding sheds where natural service occurs," Blanchard noted. "The authors’ findings encourage us not to be overzealous in treating many of these stallions that culture positive for potential pathogens."

Paradoxical Relationship Between Fertility and Stress

In the final study Blanchard described, a group from Australia performed a series of clinical trials to try to determine a relationship between stallion fertility and oxidative stress. They looked at the characteristics of dismount semen (analyzed 1-4 hours after collection); the relationship between fresh semen (analyzed immediately) and oxidative stress; and preferential oxygen requirements for stallion sperm. The researchers found that dismount sperm samples resulting in pregnancy unexpectedly had lower membrane and chromosomal integrity. These findings were associated with increased oxidative stress measurements. With the fresh semen they found that oxidative stress parameters were highly correlated with motility parameters. And, finally, they found that stallion sperm prefer (i.e., had greater motility in) an aerobic (requiring oxygen) compared to an anaerobic environment. "They concluded that stallion sperm were highly dependent on oxidative phosphorylation for energy production," Blanchard said. Simply put, the most fertile sperm "live fast and die young," he said.

"I expect this finding is probably going to result in a resurgence of research to try to identify better extenders that are more protective against oxidative stress damage to sperm," Blanchard concluded.

About the Author

Alexandra Beckstett, The Horse Managing Editor

Alexandra Beckstett, Managing Editor of The Horse and a native of Houston, Texas, is a lifelong horse owner who has shown successfully on the national hunter/jumper circuit and dabbled in hunter breeding. After graduating from Duke University, she joined Blood-Horse Publications as Assistant Editor of its book division, Eclipse Press, before joining The Horse.

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