AAEP Convention 2001: Reproduction Research
Presentations on reproduction have long been a hallmark of AAEP gatherings, and this year was no exception. The full-day reproduction session was split into two parts--the first was of a general nature, while the afternoon in-depth session involved a discussion of techniques for the use of frozen semen.
Dickson D. Varner, DVM, MS, Dipl. ACT, of Texas A&M University, reported on research aimed at determining a reason for infertility in stallions whose sperm appear to be progressively motile and morphologically normal.
One of the reasons for infertility in these stallions, Varner said, has been spermatozoal acrosome dysfunction. The acrosome fits over the head of the sperm like a bathing cap. As the sperm travel to the oviduct to meet the ovulated egg, there is an acrosomal reaction or maturational change, which is necessary for the sperm to penetrate the egg.
In some stallions, Varner said, this reaction doesn't occur and the result is a non-fertilized egg. At the moment, science can test sperm to determine whether acrosomal reaction occurs. However, because no one yet knows what causes the dysfunction, there is no therapy to solve the problem.
Still other reasons for infertility in apparently healthy and motile sperm, reported C.C. Love, DVM, PhD, also of Texas A&M, are irregularities in spermatozoal DNA and damage to it during the cooling process.
A flow cytometer can evaluate a great many sperm at one time for DNA abnormalities. Used in the process are red and green dyes. If the DNA in a spermatozoon is normal, the green dye will attach to it. If it is abnormal, the red dye will attach.
It was found that DNA from some sub-fertile stallions deteriorated rapidly after 24 hours at 5°C, the temperature to which semen is normally cooled for storage prior to artificial insemination. If the semen is stored at higher temperatures, Love said, the denaturation process of DNA is accelerated.
Testicular Size and Function
Terry L. Blanchard, DVM, MS, Dipl. ACT, also from Texas A&M, reported on an evaluation of testicular size and function in young stallions. The question was whether scrotal width was a reliable guide in determining semen quality in young stallions. Results of 53 breeding soundness examinations of 3-year-old stallions of eight breeds were compiled, and that research demonstrated that scrotal width was only moderately correlated with parameters of semen quality in first and second ejaculates.
Equine practitioners should be cautious about determining the number of mares to be bred by a young stallion based only on scrotal width, Blanchard said. The stallion's semen should be evaluated before making such a determination.
Of the 53 stallions evaluated in the breeding soundness examinations, 25% had unsatisfactory semen quality.
In another part of the study involving testes from a slaughterhouse, Blanchard said he was surprised to find that the horse's age--ranging from one to three years--had little to do with the weight of the testes.
The take-home message was that it is very difficult to make predictions about sperm quality and number based only on testicular size.
Marbles in Mares
Gary Nie, DVM, MS, PhD, Dipl. ABVP, formerly of Auburn University and now of the Rood and Riddle Equine Hospital in Kentucky, offered a surprising presentation. He discussed the effects of placing a glass ball or marble in a mare's uterus to suppress behavioral estrus.
Nie said he learned about the procedure online, where it was described by a veterinarian in the Netherlands. The procedure, he said, involves placing a 35-millimeter marble in the uterus within 24 hours post-ovulation. When the mare is in heat, Nie said, the ball can be slipped into the uterus through the cervix. Removal is facilitated when the mare eventually returns to estrus. At that time, the ball can be manipulated to the cervical opening and easily removed.
The goal is to prevent estrus in mares without resorting to drug therapy and risking potential harmful, lasting side effects.
Use of the glass ball, he said, was successful in preventing 40% of the mares in a study from coming into heat for three months. "At this time," Nie said, "it's unclear why or how this occurs."
It also appears from early evidence that once the ball is removed, the mares will cycle normally without any reproductive impairment.
Nie said that it is important to use a 35-mm ball because when 25-mm balls were used, they were frequently expelled.
During the afternoon reproductive session, speakers discussed the sometimes frustrating use of frozen semen. The cattle industry has mastered the procedure and has reported high success rates with frozen semen; however, the same has not been true with the horse industry. At best, equine pregnancy rates are lower when frozen semen is used as compared to fresh and cooled semen, and at worst, the semen of some stallions simply can't withstand freezing.
Leading off the session was Bo G. Crabo, DVM, PhD, Professor Emeritus at the University of Minnesota, who provided a history of artificial insemination and discussed the physiological aspects of stallion semen cryopreservation.
He said the first report of artificial insemination (AI) is a story of Arabs stealing semen from a stallion owned by a rival tribe and bringing home the semen in a sponge.
The Russians, he continued, were the pioneers in AI during the early 20th Century. They were more interested in AI in horses than in cattle.
The freezing of semen in glycerol came about by accident. "In the late 1940s," Crabo said, "Christopher Polge, working with chicken and the economically more important bull semen, pioneered development of frozen-thawed semen technology. This, however, was done by unknowingly using a bottle containing glycerol and allowing the semen to remain in glycerolated extender for 18 hours. Thus, frozen semen technology was developed without much knowledge of the physiology of spermatozoa or the events leading to fertilization."
Crabo then presented information on the physiology of sperm and the freezing procedures currently being employed.
Next at the podium was Paul R. Loomis, MS, who operates Select Breeders Service, Inc., with headquarters in Maryland. His company is involved in worldwide shipment of equine semen.
"Proper handling and maintenance of semen frozen and stored in liquid nitrogen," he said, "is critical to the success of artificial insemination with frozen semen."
Loomis offered these tips in the handling of frozen semen:
- Don't allow liquid nitrogen to contact skin, as this can cause extreme frostbite almost instantaneously.
- Don't allow objects cooled by liquid
nitrogen to touch bare skin, as they might stick to the skin and tear away tissue when the object is removed.
- Don't seal containers tightly. Nitrogen containers must be adequately vented to prevent buildup of gas pressure, which can severely damage or burst the container.
- Avoid spilling or splashing nitrogen when transferring it from container to container.
- Never use a hollow tube or rod to measure liquid levels, as the gasification and expansion of the rapidly warming liquid inside the tube will force liquid to spurt from the top of the tube.
- Liquid nitrogen should be disposed of only in outdoor areas by pouring it slowly onto the ground where it can evaporate into the air.
Assessing Sperm Quality
James Graham, PhD, of Colorado State University, said that many attributes other than motility are important to sperm health and fertility. "If we look only at motility," he said, "we'll overestimate fertility."
Available to the practitioner are laboratory assays that can help pinpoint infertility problems, including the following:
- Computer-assisted sperm analysis systems that permit the accurate evaluation of not only the percentage of motile sperm, but also percentages of progressively motile cells and the velocities of the motile sperm.
- Fluorescent stains that provide greater contrast between "live" cells and "dead" cells. "Recent developments in flow cytometry and fluorometry make viability assessments rapidly and inexpensively."
- Fluorescently labeled stains that can be used to visualize the stallion sperm acrosome and assess whether it is capable of functioning normally.
- Light microscopic evaluation of stained or unstained sperm that can be used to assess sperm or sperm cells to determine whether they are normal.
- In vitro assays to determine the ability of spermatozoa to undergo capacitation and the acrosome reaction.
Graham concluded his presentation by saying, "Laboratory assays have been developed that evaluate multiple sperm parameters on large numbers of sperm in a semen sample. These assays may prove useful in evaluating semen prior to insemination or at the beginning of the breeding season. They will also be invaluable in developing new techniques for preserving stallion semen.
"Although the ability of these assays to predict fertility has not been established, recent data indicate that utilizing several assays that evaluate different attributes of the spermatozoa can be highly correlated with stallion fertility."
Elizabeth Metcalf, MS, DVM, Dipl. ACT, a private practitioner in Oregon, discussed the hurdles that must be cleared in importing and exporting frozen semen to and from foreign countries.
Rules and regulations vary country by country, she said, and involve extenders as well as semen. She discussed diseases that can be transmitted via contaminated frozen semen, such as contagious equine metritis (CEM), equine herpesvirus type 3, equine arteritis virus, equine infectious anemia, dourine, and piroplasmosis. Hemospermia (contamination of semen with blood) can be a source of blood-borne infection.
The United States Department of Agriculture (USDA) has designed a separate set of requirements based on the CEM status of the country of origin, Metcalf said. "The testing procedures for CEM are rigorous, thorough, and expensive. The only other disease specific for equids for which the USDA requires testing is dourine."
However, the USDA places strict requirements on the use of certain products in semen extenders. Milk products must originate from countries that are recognized to be free of foot and mouth disease. Eggs used in extenders must originate from flocks that are certified to be free of Newcastles disease.
"The first step to importing semen is, of course, to verify the requirements and ensure that the facility that freezes the semen is in compliance with U.S. regulations," Metcalf said. "Semen must be collected and processed under the supervision of a veterinarian. In order to clear customs regulations, a federal agent must inspect the liquid nitrogen container carrying the frozen semen."
When it comes to exporting frozen semen, Metcalf said, there is great variability among countries. The requirements might vary from simply requiring a permit to several months of quarantine for a stallion or semen.
"The individuals that are responsible for processing and using the semen," she said, "must develop an awareness of potential disease transmission and often impose even stricter sanctions than required by the respective governments."
Ovuplant Table Topic
Patrick McCue, DVM, PhD, Dipl. ACT, of Colorado State University told a packed room that a new procedure for utilizing Ovuplant was working well. The procedure, he said, involves implanting Ovuplant in the vulva rather than beneath the skin of the neck, then removing it in 48 to 72 hours. The procedure, he added, seems to have negated the problem of mares not returning to estrus after Ovuplant is used.
Breeding with Frozen Semen
Juan Samper, DVM, PhD, Dipl. ACT, of Kansas State University, discussed breeding with frozen semen. The basic concern, Samper said, involves quality.
"There is a wide variation between stallions in the ability of their semen to tolerate freezing and thawing," he said. "Rarely does the individual inseminating a mare with frozen semen have control of semen quality, and it may be suboptimal. These factors have a negative impact on the use of frozen semen in the United States. Although data are limited, it appears that (only) about 25% of stallions have first-cycle conception rates comparable with those obtained with fresh semen, even when frozen semen is inseminated into healthy mares at the proper time."
A key factor in maintaining viable sperm is using the correct thawing procedure. Semen that is packaged in 0.25-0.5-mL straws is generally thawed at 37°C for 30 seconds.
It is difficult to properly evaluate the quality of sperm after thawing, Samper said, "because there are no accepted tests that correlate well with fertility of frozen semen."
That being said, he suggested that the following guidelines be used as minimum acceptable criteria for a single insemination dose after thawing: At least 30-35% progressively motile sperm, a minimum of 50% morphologically normal sperm, and greater than six million sperm.
One of the problems faced by the equine industry, Samper said, is stallion and mare selection. "It is unfortunate," he told his listeners, "that past fertility is rarely a major criteria used by mare or stallion owners when selecting a stallion for breeding or for freezing semen. The food animal industry has shown repeatedly the importance of genetics in improving fertility."
The timing of insemination with frozen semen is critical. "Sperm that have been frozen and thawed have reduced longevity due to membrane changes that occur during cryopreservation (freezing)," said Samper. "Therefore, to maximize fertility, mares need to be bred as close to ovulation as possible."
In addition to depositing semen at the appropriate time, it is crucial that the veterinarian examine the mare post-insemination to confirm that she has ovulated and to determine whether her uterus is accumulating fluid. This exam should be carried out no more than 12 hours after insemination.
"At this time appropriate therapies, such as uterine lavage, oxytocin injections, post-breeding antibiotic infusions, and Caslick's procedures, should be performed (if needed)," he said.
It appears that breeding with frozen semen produces more post-breeding uterine inflammation than does breeding with fresh or cooled semen. The inflammatory response is normally modulated by seminal plasma. However, with frozen semen, most of the seminal plasma has been removed, and this "perhaps exacerbates the transient inflammatory response in the mare," said Samper.
Samper concluded by saying, "Frozen semen does not allow for many errors in the handling of semen and the timing of insemination. This results in an increase in labor for the veterinarian and cost for the mare owner that should be clearly discussed before starting the process. Careful selection of the mares and stallions will further increase the chances of success."
Semen Evaluation in the Field
Margo L. Macpherson, DVM, MS, Dipl. ACT, said the ambulatory veterinarian faces several challenges in field evaluation of semen, such as lack of equipment and inadequate space and means of temperature control. She described simple techniques for determining sperm concentration, motility, and the percentage of morphologically normal sperm of ejaculates collected in the field.
She listed the following necessary tools: Inexpensive bright-field microscope, hemocytometer, glass microscope slides and cover slips, pipettes or wooden sticks, slide warmer, semen extender, thermometer, eosin-nigrosin stain, imme rsion oil, small cooler, and urine sample cup.
Temperature is a critical factor when evaluating sperm motility. "Most field microscopes," she said, "will not be fitted with a stage warmer; therefore, measures must be taken to maintain semen samples at 37°C (normal body temperature) during motility evaluation. Simple tricks can be used to warm slides, cover slips, and pipettes--any square glass bottle can be filled with warm water and laid on its side to serve as a slide warmer. Warmed full fluid bags can also serve this purpose. A heating pad may be used as a slide warmer. Again, care must be taken to maintain components as close to body temperature as possible for the most accurate assessment of motility."
An examination to determine sperm morphology, she said, can be handled the following way: Use a drop of well-mixed semen with a drop of stain on a microscope slide. A second single slide is used to make a smear. Eosin-nigrosin stain is a background stain; therefore, the best slide is obtained when the smear is not too thin. The slide is allowed to dry and can be evaluated immediately or stored and evaluated later in the day.
Sperm abnormalities, she said, are optimally detected using immersion oil and 1000X magnification. Macpherson recommended that a minimum of 100 cells should be evaluated. The evaluation, she said, will yield information on normal sperm, abnormal sperm heads, detached sperm heads, abnormal midpiece shape, coiled tails, and premature germ cells.
Breeding Dummy Use
Sue McDonnell, PhD, who heads the Equine Behavior Lab at New Bolton Center, discussed how to select and fit a breeding dummy mount for a stallion.
"Probably the most important aspect of fitting the dummy mount is height," McDonnell reported. "A good starting height for most normal, physically able stallions of average body length is estimated by having the top of the back of the dummy set to the height of the tailhead of the stallion. It can be adjusted slightly lower or higher for best performance."
McDonnell shared these observations concerning general breeding dummy design features:
- A single, stout, "clean-cut," padded pedestal reduces risk of entanglement and serious injury during awkward dismounts or falls.
- A smooth, snug-fitting cover made of a "cool" material with no wrinkles, bulky seams, exposed fittings or laces minimizes the risk of rub sores to the knees and chest, and also minimizes penile injuries.
- Modest padding will soften the mount and dampen the sound of the stallion hitting the dummy mount.
- Sturdy construction without rattles and clunks and the associated movement as the stallion mounts considerably improves efficiency of training for many stallions.
- The position of the dummy within the breeding space is important. In general, stallions do best with ample front and side clearance and ample head room.
- Good footing can enhance a stallion's trust and ease of mounting and dismounting.
- Built-in grooves can enhance the stallion's ability to lock in the forelegs for breeding.
- The angle of the barrel, whether level or at a slight angle sloping up from the tail to the head, does not appear to be an essential feature if the dummy height at the tail is otherwise well-fitted to the stallion.
- Accoutrements, such as a mane, tail, or head to make the dummy more natural-looking, generally are unnecessary.
- Self-service dummies with a built-in vagina can work well for some stallions. (However, McDonnell warned, penis injuries and behavior problems are probably more common with self-service dummies than for those requiring hand-held vaginas.)
- Adjustable-height or -angle dummy mounts are also useful, but are difficult to use without problems of movement and rattle.
- "Stretch limo" dummy mounts, with barrels longer than six to eight feet, can be used to accommodate stallions which are tough to hold squarely in normal breeding position.
About the Author
Les Sellnow is a free-lance writer based near Riverton, Wyo. He specializes in articles on equine research, and operates a ranch where he raises horses and livestock. He has authored several fiction and non-fiction books, including Understanding Equine Lameness and Understanding The Young Horse, published by Eclipse Press and available at www.exclusivelyequine.com or by calling 800/582-5604.
POLL: University Equine Hospitals