Frozen Semen

Frozen Semen

Frozen Semen Manual

The use of cryopreserved semen in the equine breeding industry is growing in popularity. Mare owners are now able to choose a stallion anywhere in the world to which to breed and stallion owners can breed more mares without disrupting their stallion's schedule. Proper equipment and knowledgeable veterinarians are needed for the intensive management required for success with frozen semen. Veterinarians need to be competent not only in the management and insemination of mares, but also in handling and evaluating frozen semen.

Introduction to Frozen Semen

A great deal of variability exists among stallions in the ability of their semen to withstand freezing and thawing. Equine semen is far less tolerant of the process than bull semen; no single technique or formula exists to successfully freeze all equine semen. Semen from some stallions fares better than others with certain freezing media. Also, tolerance of the freezing and thawing process may show some variation within an individual stallion for different ejaculates and at different times of the year.

It is generally accepted that frozen/thawed semen does not maintain viability within the mare for as long as fresh or fresh cooled semen. Whereas the number of progressively motile sperm needed for optimal pregnancy rates using fresh semen has been established as 500 X 106, the minimum breeding dose to optimize pregnancy rate is unknown for frozen semen usage. Insemination with frozen/thawed semen is performed very close to ovulation and dosages of 200 X 106 progressively motile spermatozoa (post-thaw) have obtained acceptable pregnancy rates. Dosages less than this may compromise fertility.

Predicting Fertility

Semen from some stallions may appear to freeze exceptionally well, yielding postthaw motility rates of >60%,and yet few, if any, pregnancies are achieved. For other stallions, careful management of frozen/thawed semen with questionable motility and character has resulted in excellent pregnancy rates. It therefore seems reasonable to assume that motility may not be the best predictor of fertility of frozen semen.

Currently, many researchers are examining other means of predicting and assessing fertility of the stallion. The best means to predict a particular stallion's fertility with frozen/thawed semen is to review his breeding history, including the postthaw motility and character of the semen as well as his pregnancy rate per cycle. If this information is unavailable, obtain information on the stallion from the person who froze the semen. Currently, a semen certification program for individuals freezing equine semen is being developed, to enhance and ensure continuing excellence and education in this industry.

Pregnancy Rates

For some stallions, first-cycle pregnancy rates are lower with frozen semen than with fresh cooled semen. Acceptable first cycle pregnancy rates ranging from 31-73% have been reported, with the highest rates achieved when mares of known fertility were examined every 6 hours with transrectal ultrasound, administered hCG and inseminated just prior to and directly after ovulation. In addition, higher success rates were obtained using frozen semen from stallions of proven fertility.

In recent years several studies have revealed no significant difference in foaling rates between mares that had been inseminated with extended semen immediately following collection, extended semen cooled for 24 hours or frozen semen. With continuing improvement in the freezing techniques and media, as well as the familiarity of the inseminator with the protocols and procedures, overall pregnancy rates should continue to improve.

Prebreeding Evaluation of the Mare

A thorough breeding soundness exam can aid the practitioner in evaluating the suitability of the mare for breeding. This examination may include, but is not limited to:

Historical Evaluation-including age, parity and previous reproductive problems
Body Condition Score & Perineal Conformation
Palpation and Ultrasound Examination of the Reproductive Tract per rectum
Vaginal Speculum Exam
Endometrial Cytology
Endometrial Culture
Endometrial Biopsy

Hormonal Control of the Estrous Cycle

Hormonal manipulation can be useful in regulating the estrous cycle of the mare, either with the use of prostaglandins or progestins or both. If a mature functional corpus luteum is present on an ovary, administration of prostaglandins will bring a mare back into estrus. The interval from prostaglandin injection to ovulation will vary greatly, depending on the size and status of the largest follicle at the time of administration. Alternatively, an oral progestin, Regumate, can be administered for 7-14 days, with or without prostaglandin administration on the last day, to aid in bringing mares into estrus at a more predictable time. The most predictable results are obtained using Progesterone & Estrogen (P&E).

Ovulation and the use of hCG

Once the mare is in estrus, continuing examination of the reproductive tract should consist of both palpation and ultrasonography. Follicles should be evaluated for size, shape and consistency and the uterus evaluated for endometrial folds and presence of intraluminal fluid.

The number of doses of frozen semen available many be limited in many instances.

Although there is no question that a mare should be bred as close to ovulation as possible, there is controversy as to whether to breed before or after ovulation. It is generally accepted that inseminating immediately prior to or just at ovulation will result in the highest pregnancy rate. From a management point of view, postovulation breeding is far simpler since it is easier to detect a corpus hemorragicum by ultrasound examination than it is to accurately predict impending ovulation . The ideal regimen would be to inseminate just prior to ovulation and just after a corpus hemorrhagicum is detectable by ultrasonography.

Administration of a GNRH anolog or hCG (2500-5000 iu), when a softening, preovulatory follicle is >35mm and uterine edema are detected, results in ovulation within 36-44 hours. Consequently, administration of hCG in the morning and insemination the following evening may work well in most instances. In the interim, the mare should be examined every 8 hours to detect an earlier ovulation. If the mare has ovulated, she should be inseminated immediately. If the mare does not ovulate after 36 hours, she should be examined every 6-8 hours and inseminated every 12 hours prior to ovulation and once immediately following ovulation.

Preparation of the Mare for Insemination

The mare should be prepared for insemination before the semen is thawed. If possible she should be restrained in stocks. Her tail should be bandaged and tied away from contact with the vulva and perineum. The vulva should be washed well with liquid soap and rinsed thoroughly with water. The vulva and perineal area should then be dried with a clean paper towel.

Handling the Frozen Semen

The semen is stored in a liquid nitrogen (LN2) tank at -196o C. The tank may contain LN2 in an aqueous state at the bottom of the tank or the tank may be a vapor shipper that has been primed with LN2. The LN2 is absorbed into the inner lining of the tank which continually emits N2 vapor that will maintain the semen at -196o C.

Your veterinarian will read all instructions which accompany the shipment of semen before thawing any semen for insemination. It is imperative that the stallion is properly identified. This information will ascertain how many units (straws) constitute a single insemination dose. More than one insemination dose may be sent, and more than 1 straw may consitute an insemination dose.

Semen may be packaged in straws (0.25-5 ml), pellets in whirlpack bags, or plastic or aluminum packets. The smaller straws may be free in the canister, surrounded by cotton, or resting in plastic goblets, which may in turn be clipped to metal canes. Most metal canes hold 2 goblets. Straws may be easily identified while in the vapor by their color or the colors of the glass bead at the top. Straws are never removed until we are ready to transfer them to another tank or a water bath for thawing.

Thawing Procedure & Evaluation of Sperm Motility

The thawing procedure is a critical step in the fertility of the semen for which there are a number of different methods. The number of sperm in the insemination dose can be counted with a hemacytometer using semen not yet mixed with an extender which is diluted using a Unopette blood cell diluent system.

Postovulatory Examination

Mares should be examined following the last breeding for the presence of fluid in the uterus. There is a physiological endometritis that ensues following all inseminations; a normal mare clears the inflammation quickly. Frozen semen tends to enhance this endometritis and in some mares, it is quite severe. Veterinary intervention in the form of warm saline (buffered) lavage, antibiotic infusions, and exogenous oxytocin administration, etc. may be warranted. These procedures should not be conducted until 4-8 hours following insemination.

This article has been slightly modified by East Coast Equine Reproduction Center for the purpose of client education.

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