Researchers investigate new ways to improve fertility in mares and in stallions

by Denise Steffanus

BREAKTHROUGHS in treating placentitis, the role of certain seminal proteins in conception, and links between blood flow to testicles and sperm production lead this year's research projects for reproductive health. And an ancient remedy, acupuncture, is gaining acceptance in treating mares with reproductive problems.

At the University of Florida's large-animal reproduction facility, Margo Macpherson, D.V.M., M.S., associate professor of theriogenology, is exploring the use of drugs to treat mares with placentitis and other high-risk pregnancies.

New data has shed light on how drugs are transported across the placental barrier, which gives researchers another tool in fighting placentitis.

"We previously thought that maybe they did not pass the placental barrier, but we know that they do that now," said Mats Troedsson, D.V.M., Ph.D., chief and professor of theriogenology at the University of Florida. "There are some clinical trials run with very good results of combined treatment of Regu-Mate, antibiotics, and anti-inflammatories, and that really shows that we can save many of these pregnancies."

Troedsson said the next challenge is to identify which pregnancies can be saved using the combination drug therapy and which cannot.

Proteins key to fertility

Troedsson also has been studying the role of certain proteins produced in the stallion and their effects during the interaction of sperm with the uterus. He believes these proteins form a healthy environment for sperm cells and that any abnormality in that protein environment may affect fertility.

"In order for the sperm cells to be able to bind to an egg and fertilize an egg, they have to have some proteins in the membranes," he said. "Those proteins often come from epididymal fluid, fluids in the testicles during [sperm] production, and some of them come from the accessory sex glands. We can measure the concentrations of those proteins in seminal plasma and maybe use that as an indicator for some problems with stallions."

Troedsson recently identified one of these proteins that has significant involvement in the interaction between sperm and the uterus. The finding was the impetus for Troedsson to apply for a grant from the Grayson-Jockey Club Research Foundation to pursue the course of investigation.

"The breakthrough is that if we identify specific proteins with biological activity, then we have something to measure, and we can then correlate that to fertility," Troedsson said. "We don't have anything really that correlates very well to fertility other than how many sperm cells we can see moving around, which is a very rough way to measure fertility."

Troedsson said the work could benefit the Thoroughbred industry, even though it is limited to live-cover breeding.

"If a stallion is deficient in that protein, even in the Thoroughbred, we could actually add that protein to a semen extender and then infuse the semen extender [in the mare] prior to breeding," he said. "That means that when the semen gets into the uterus, it will mix with the extender to restore a healthy makeup of proteins. So that is the goal of our proposal."

Acupuncture for fertility

Acupuncture has been used for years to aid women's reproductive health. Now, that technology has emerged in equine medicine to solve the problem of uterine fluid in the mare and to promote ovulation.

In some mares, poor reproductive conformation may cause urine to dribble down the vulva, pool inside the vagina, and even seep into the uterus, causing infection. Other mares may develop inflammation when they cannot clear fluid from the uterus after being bred. Typically, mares that accumulate fluid are difficult to get in foal or, if they do conceive, they seldom maintain the pregnancy.

Rhonda Rathgeber, D.V.M., Ph.D., a field-care veterinarian at Hagyard Equine Medical Institute in Lexington, is using acupuncture to treat mares with this problem. A certified veterinary acupuncturist, Rathgeber employs electroacupuncture to stimulate the nerves that supply the uterus. Electroacupuncture differs from traditional acupuncture in that once the needles are inserted into the proper acupoint, wires are clipped onto the needles to connect them to a device that delivers minute electrical pulses.

"The clinical problem is that [these mares] cannot clear the fluid from the uterus post-breeding," Rathgeber said. "The electroacupuncture stimulates the dorsal nerves that supply the uterus to increase tone ... and when the uterus is more toned, it sits up higher, and the mare is able to expel that fluid."

Rathgeber said it takes only 24 to 48 hours after treatment for the difference in uterine tone to be palpable. She believes electroacupuncture is becoming a common treatment for fluid pooling because treating veterinarians are able to detect results the next day.

"Unfortunately, no controlled studies have been done to measure the diameter of the uterus through ultrasonography or to measure muscle activity in the uterus after treatment," Rathgeber said.

"I'd like to put together a study. Dr. Troedsson down at Florida has a mechanism to measure smooth-muscle activity in the uterine wall, which would be very interesting to do post-acupuncture to see what kind of changes you get."

Rathgeber said no funding is currently available for such a study, but she hopes money will be earmarked sometime in the future to substantiate the validity of the treatment.

Follicle production

Rathgeber also uses acupuncture to raise levels of follicle-stimulating hormone (FSH) in mares that do not cycle properly or produce a follicle each month.

One of the theories of how acupuncture works is called the humeral mechanism, she explained. Through the humeral mechanism, acupuncture raises levels of certain hormones and other substances circulating in the blood. When acupoints that trigger these humeral responses are stimulated by traditional tai-chi needles or injections of vitamin B-12, FSH levels rise, as well as endogenous progesterone, which is essential for maintaining a pregnancy.

Rathgeber said acupuncture treatment also is good for reducing any kind of inflammation within the reproductive tract that may impede conception or cause abortion.

"It's used quite a bit for maintaining pregnancy in women who have had multiple miscarriages, so I've started to do horses that have had multiple abortions," she said. "Year after year, they just can't seem to hold a pregnancy to term. The past few years, I've tried to treat a few of those each year. And there's something to it. There seems to be increased endogenous progesterone with it. And, obviously, you're trying to reduce any inflammation in the uterus or in the placenta."

Studies in cattle have proved the validity of this theory of acupuncture-induced humeral mechanism, she said.

"If you do acupuncture on one animal and pull blood from that animal and put it into a second animal, you'll see some of the effects of acupuncture [in the second animal]," Rathgeber said. "So there is quite a bit of research to say that humeral substances in the blood are released, and how acupuncture works is in releasing those endorphins."

Rathgeber recommends acupuncture as a first-line treatment for fluid pooling and for mares that are not cycling properly. But she acknowledged that clients at first were apprehensive about it as a first-line treatment.

"Originally, when I first started doing it six or seven years ago, it was the last stretch," she said.  "They'd say, 'We can't get her in foal. Can you fix her?' "

Now, Rathgeber's clients are more receptive to the treatment.

"Usually, the clients that I have have gotten used to using it now, and they will call for it first," she said.

For problem mares expected to head to the breeding shed in February, Rathgeber advocates beginning acupuncture treatment in January.

"It depends totally on the case," she said. "If the mare has had a history of problems, I would start in January or February with her. If the mare is healthy, young, and not having a history of any problems, I would just do the routine veterinary [work] and then try to breed her one or two times. Then, if you're having problems, do something with acupuncture. I don't think every single mare needs to have acupuncture before the season.

"Don't wait until you've been to the shed five times, or if there are obvious problems, or if the mare has chronic uterine fluid seen via ultrasound. Those would be good cases for it, or mares that you just can't get to cycle."

For mares that have a problem with pooling fluid, Rathgeber said the number of treatments the mare should have depends on the individual case.

"It depends on how much fluid she has, if she was treated the last cycle, and where she is in her cycle," she said. "Sometimes, I just get one or two treatments in them per breeding; sometimes mares that have chronic problems that I have treated year after year, I try to get one or two treatments in them before breeding and one or two treatments after breeding."

Each treatment costs between $100 and $200, with electroacupuncture used for fluid pooling costing about $175 per treatment.

Rathgeber emphasized that owners who are interested in obtaining acupuncture treatment for their horses should solicit the services of a veterinarian certified in veterinary acupuncture. She added that it is essential for this individual to work closely with the owner's reproductive veterinarian to achieve the best results for problem mares.

The best candidates for acupuncture are young mares with fertility problems. "You get into other problems with the older mares," she said.

In a presentation on the subject at the 2006 Bluegrass Equine Symposium in Lexington in October, Rathgeber said: "Reproductive conditions treated commonly with acupuncture include anestrus, retained corpus luteum, urine pooling, uterine fluid, vaginitis, abortion, dystocia, retained placenta, uterine prolapse, cryptorchidism, and decreased libido in stallions."

Blood flow to testicles

Malgorzata Pozor, D.V.M., Ph.D., assistant clinical professor of theriogenology and a member of the University of Florida's large-animal reproduction faculty, is conducting ongoing research to see if blood flow to the testicles correlates with sperm production in breeding stallions.

Pozor employs a form of ultrasonography called spectral Doppler to measure the velocity of blood flow to the testicles through a noninvasive procedure.

"Using color Doppler ultrasonography, you can accurately evaluate blood flow," Pozor said. "Because the blood cells are moving in a tube [blood vessel] in one direction and the ultrasound beam goes parallel to it, there is a Doppler shift--changes of frequency of the ultrasound wave--that depends on the velocity of the blood moving in the vessel. In this way, not only do you visualize the movement of blood, but also you can objectively measure it. You can do this based on the intensity of the color, but also you can look at a graph that will show you a very detailed distribution of the velocity of blood cells moving in the vessel. So it's a very precise method. You can see pulsation and distribution of blood vessels within the organ."

Pozor said that although the technology has been well accepted in human reproductive medicine, it has not been well proven in the horse, which is the goal of her current studies.

"What we want to see is if there is a correlation between the quality of semen and testicular blood flow, and that's going to take more time and more science," she said. "But, also, we would like to affect blood flow medically to try to improve testicular perfusion [blood saturation]."

If this research returns the results Pozor expects, the procedure could be used to diagnose pathologies in the testicles, to monitor the efficacy of various treatments to improve blood flow to them, and to determine if there is a way to enhance blood flow to the testicles that would increase sperm production and quality.

Pozor said she hopes to have preliminary results of her study in 2008.

In previous studies, Pozor and her colleagues have shown that blood flow to the testicles increases during breeding season and is correlated with an increase in concentrations of testosterone.

"This appears to be primarily from an increase in the diameter of testicular artery and increase in the blood-flow mean velocity," she wrote.

"This is something that could be very useful in the Thoroughbred industry," Troedsson said of Pozor's work.

Denise Steffanus is a contributing editor of Thoroughbred Times who writes frequently on veterinary and farm management topics.