To reduce time and expense involved in breeding a mare with cooled transported semen, the practitioner should strive to minimize the number of shipments required for breeding. If pregnancy does not result from breeding during one estrous cycle, expenses are multiplied as additional breeding(s) will be necessary. If breeding during two estrous cycles does not result in pregnancy, a thorough breeding soundness examination (if not already performed) may be necessary to determine if the mare is at fault.

The practitioner should play a primary role in maintaining quality control (semen quality, insemination dose, timing of breeding, etc.) when horses are bred with transported cooled semen. To be successful, stallion owners/managers, mare owners, and veterinarians involved in stallion and mare management must work together as a team to optimize the chance of conception from each breeding.

Prebreeding preparation of the mare

Prior to the onset of the breeding season, the mare should be examined to detect reproductive abnormalities. Underlying problems that might contribute to infertility, such as poor body condition or endometritis, should be corrected.

Prediction or detection of ovulation

When the mare is in estrus, the cervix becomes progressively more relaxed and the uterus becomes softer and more edematous as follicle size increases and ovulation approaches. Once a follicule reaches 35-50 mm in diameter, the ovulatory follicle(s) soften(s) detectably on palpation per rectum. Using transrectal ultrasonography, the cross-sectional size and shape of follicles and the echogenicity of the follicular fluid can be used to aid in prediction of ovulation. Within 24 hours prior to ovulation, the shape of most follicles tends to change from spherical to a conical or "pear" shape, and the follicular wall may become "scalloped" or thickened in appearance. The apex of the conical follicle will be located at the ovulation fossa. The echogenicity of the follicle changes from a predominantly anechoic appearance to a heterogenous echotexture as ovulation occurs and luteal development begins. The developing corpus luteum often contains a small anechoic center resulting from retained follicular fluid or blood.

Hormones to control ovulation

When cryopreserved semen is used for breeding, or when breeding to certain subfertile stallions, the fertilizable lifespan of sperm may be reduced, requiring breeding closer to the time of ovulation. To optimize fertility, hormones can be adminstered to increase the probability that ovulation will occur near the time of breeding with cooled semen. Onset of estrus can be controlled in cycling mares by administration of progestogens and/or prostaglandins.

Human horionic Gonadotropin (hCG) or a GnRH analog can be administered to reliably induce ovulation. Administration of hCG (1500-3300 units, i.v., or i.m.) when a 3 35 mm in diameter follicle is present in mares in estrus will induce ovulation in a majority of mares within 36-48 hours. If the day of estrus is unknown, and the follicle is larger than 35 mm in diameter, the interval to ovulation is less precise and may occur earlier than expected after hCG administration. Some practitioners believe hCG will more predictably induce ovulation at a precise time if endometrial edema (as detected by ultrasound examination) is maximal at the time of administration.

The stallion manager should be consulted to make sure semen will be available for breeding when the mare is expected to be in estrus. Human chorionic gonadotropin (hCG) can also be used to improve synchrony between arrival of shipped semen and time of ovulation. When a mare is in estrus, daily or even twice daily examinations are performed to monitor size of developing follicles. Typically, once follicular diameter reaches 35 mm, hCG may be administered after it is confirmed that transported semen can be obtained and will arrive the next day. The semen generally arrives 24 to 36 hours later in time for breeding just prior to ovulation.

Timing and frequency

Since viability of stallion semen may be shortened by cooling, many practitioners recommend breeding as near to ovulation as is feasible to optimize pregnancy rates. While satisfactory pregnancy rates have been reported in mares inseminated after ovulation with transported cooled semen, critical study of the optimal time for breeding in relation to ovulation with cooled semen has not been done.

Breeding the mare with cooled semen

When the cooled semen arrives for insemination of the mare, current recommendations are to:

Overseas workers often recommend warming the cooled semen to body temperature prior to insemination of the mare. In the U.S., the cooled semen is usually inseminated directly into the uterus as soon as possible after opening the shipping container. Since no detrimental effects on pregnancy rate have yet been determined by inseminating mares with cooled semen, at present we see no reason to prewarm the cooled semen prior to insemination unless a cream-gel extender is used. This extender forms a gel at refrigerated temperature, so it must be warmed prior to insemination. The packaged sample can be placed in an incubator or waterbath preset at 37-38oC. It is important to avoid contamination of the extended semen with water when warming the sample in a waterbath.

Double inseminations

It has become common practice for many stallion owners/managers to prepare two bags (insemination doses) of extended semen for shipment - one to be used for an initial insemination upon arrival, and one to be held for insemination again the next day. For many stallions, the longer the semen is held at refrigerated temperature, the poorer sperm motility becomes. However, if semen is to be held for breeding again the next day, precautions should be taken to maintain the cooled semen at 4-6oC until the time of the second insemination. Once a shipping container is opened for the first breeding, temperature of the semen left in the container is likely to rise above 10oC, causing premature sperm death. The remaining semen dose can be immediately placed in an insulated box within a refigerator (4-6oC) - until it is to be opened for breeding the next day. This would be especially critical for semen cooled in some "disposable" type shipping containers which may only maintain semen at < 10oC for 27.5 to 33.5 hours.

Postbreeding treatment for mares with intrauterine fluid accumulation

If a given mare needs treatment after breeding that includes evacuation of uterine fluid or debris, we recommend either postbreeding uterine lavage or oxytocin treatment. Treatment as early as 4 hours after breeding does not appear to affect fertility. Treatment can also be performed after ovulation is detected. Intrauterine treatment more than 2 days after ovulation should be avoided since it may impair CL function, resulting in an early return to estrus with subsequent loss of pregnancy if the breeding was successful.

Examination for pregnancy

We recommend the mare bred with transported cooled semen be examined for pregnancy using transrectal ultrasound at 14-15 days post-ovulation. This enables early detection of a pregnancy that is normal for stage of development, allows early detection of twins before they become fixed in the uterus, and provides time for rebreeding during the same estrus if the mare failed to become pregnant.