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Frozen Semen

INTRODUCTION:

    • Frozen Semen for artificial insemination (FAI ) has been used in dairy cattle successfully for years.
  • However it historically has suffered a slow acceptance in the horse industry due to breed association restrictions and poor pregnancy rates early in its development.
  • The protective action of glycerol in the freezing of semen was discovered by accident in 1949.
  • Since then most of the research and funding has been in the freezing of bull semen.
  • The first successful recorded equine pregnancy using FAI was not until 1975.
  • FAI has been used extensively in other parts of the world in recent years. China inseminated 110,000 mares between 1980 and 1985 and claimed a 68% pregnancy rate.

ADVANTAGES OF FAI

  • Economics
    • It is cheaper to ship frozen semen across country than it is to ship a mare.
    • Decrease cost of mare care by keeping the mare at home
  • There is less separation anxiety for owner.
  • There is less stress and exposure of the foal to disease when he stays at home.
  • A stallion can continue to be used for competition during the breeding season or if he becomes injured or ill, his breeding season can continue through the use of FAI.
  • Semen can be stored indefinitely for future use.
  • The differences in breeding season between the northern and southern hemispheres would not be a problem.
  • The use of genetically inferior stallions should decrease.
  • Interest in breeding and raising horses should increase with the access to superior stallions that FAI provides.

DISADVANTAGES OF FAI

  • Not all stallions are good candidates for FAI, approximately only 30% of randomly tested stallions have good pregnancy rates with frozen semen.
  • Poor pregnancy rates are still a problem.
  • Some breeders are concerned that the gene pool will be decreased by many mares being bred to a selected few stallions. For most stallions, the average amount of mares that can be bred in a year totals only 200-300 with FAI. There doesn’t appear to be great danger of the gene pool shrinking and in fact FAI may expand the available gene pool by making overseas stallions available.
  • The income of some stallion owners will decrease.
  • Stud fees may decline. Some mare owners may consider this an advantage. The stud fees will not drop as dramatically as with bull semen because of the high cost of collecting, processing and storing equine semen. The cost of collecting and preparing enough semen for one pregnancy is approx $150.00.
  • Errors can be made in the identification of the offspring from frozen semen. However, the frozen semen straws are clearly marked and the stallion is indicated on each straw. The identity of the foal and the integrity of the pedigree are determined by the breeder, not the method of breeding. If there are questions the parentage can be confirmed by blood tests.

STALLION CONSIDERATIONS / SEMEN QUALITY

    • The stallion must be physically able and trained to use the artificial vagina for collection of semen.
    • It is also beneficial if he is trained to the phantom for easier collection.
    • The semen quality after thawing will vary between stallions:
    • – 30% of randomly tested stallions will have 80 % of the pre freezing motility.



      – 30% of randomly tested stallions will have 50% of their pre freezing motility.



  • Post thaw motility can vary between ejaculates from same stallion.
  • Good post thaw motility doesn’t insure good fertility. Cold shock during freezing can damage the spermatozoa’s membrane structures and interfere with fertilization.
  • Semen motility is determined by microscopic exam and is notoriously imprecise. Studies have shown that visual evaluation of semen motility tends to over estimate percent semen motility. The most accurate means of evaluating equine semen motility is the use of computer programs.


On average, 35% of stallions are classified as good prospects for frozen semen on basis of

1. Pre freezing motility > 60%
2. >70% of spermatozoa are morphologically normal.
3. >30% post thaw motility
4. Some spermatozoa activity for 120 hr. post thaw, stored at 1 degree Cent.

  • 25% of stallions are classified as average candidates for freezing semen
  • 40% of stallions are classified as poor candidates for freezing semen.
  • Stallions rated good by this method also tended to be more consistent between ejaculates, producing acceptable post thaw samples in 70% of the ejaculates, whereas average rated stallions produced acceptable samples from only 50% of the ejaculates

An acceptable semen sample should have 35% post thaw motility and 200 million progressively motile spermatozoa.

PREGNANCY RATES

There are many conflicting reports of pregnancy rates obtained with FAI. The reports list if the mare got pregnant or not, however this is not always an accurate assessment of the stallion or the breeding method being studied. There are other considerations such as:

1. How many cycles was the mare bred?
2. What was the fertility status of the mares used in the study?
3. How many spermatozoa per insemination dose?
4. How close to ovulation was the mare inseminated?

On average, a good freezing stallion should achieve about 60% of his live breeding pregnancy rate when frozen semen is used, i.e. If a stallion has an 80% fertility rate on one cycle with live cover and he is a good freezing stallion he should get 48% of the mares bred with frozen semen on the first cycle.

Stallions can vary in pregnancy rates between ejaculates. Pregnancy rates for one stallion can range from 8% to 65% depending on which ejaculate is used.

The average pregnancy rate for one cycle of breeding is approximately 25-30% and 45-50% for the entire breeding season. TIMING OF INSEMINATION

  • Better results when insemination occurs close to ovulation. T
  • The freeze thaw cycle damages the membrane of the spermatozoa, causing partial capacitation and an inability to attach and store viable sperm cells at the caudal isthmus portion of the oviduct.
  • Frozen semen typically has short livability…6-8 hours
  • Pregnancy rate drops dramatically when insemination occurs >24 hours of ovulation
  • The mare should be inseminated within 12 hr pre and post ovulation.

EARLY EMBRYONIC LOSS

  • Some studies indicate a higher incidence of early abortion when frozen semen is used.
  • Normal loss rate is approx. 15 %.
  • Loss rate with frozen semen is reported to be 21%.

PRE BREEDING CONSIDERATIONS

When selecting a mare to breed with frozen semen, the following factors should be considered:

1. Age – mares > 12 years old suffer a significant decrease in fertility.
2. Breeding history – mares that have been previously diagnosed with uterine pathology are poor candidates.
3. Mares with foals have a slightly higher success rate than maiden mares.

Once a mare has been selected as a candidate for FAI a pre breeding exam should be done. Special consideration should be given to:

1. Vulvar conformation – poor conformation will predispose to infection and infertility or abortion.
2. Ultra sound exam of ovaries/uterus/cervix checking for anatomical abnormalities, uterine tone, cervical tone, and ovarian activity.
3. Vaginal exam- checking for urine pooling, cervical scars, cervical discharge
4. Uterine culture / cytology
5. Biopsy of uterine mucosa may be helpful if there are some questionable findings or history.
6. Ultra sound exam of ovaries and uterus to look for ovarian abnormalities, uterine cysts or fluid in uterus.

BREEDING CONSIDERATIONS

  • Strictly adhere to instructions for handling semen supplied by stallion owner.
  • Estimation of time of ovulation requires rectal exam done manually or with an ultra sound.
  • Signs of impending ovulation are:
    • Increased heat score,
    • Softening and open cervix,
    • Uterine folds / follicle. On ultra sound, the follicle reaches maximum size (43- 47 mm) and becomes distorted in outline from spherical to irregular and the uterine folds change from the cartwheel like in appearance with dark tips seen in mid heat to uterine folds that are less edematous.

Equipment needed for actual insemination with frozen semen:

1. Sterile sleeves
2. Non -spermicidal water base lube
3. Haver Lockhart AI pipettes
4. Air Tite syringes
5. Water bath (usually 37.5 degree centigrade)
6. Forceps for removal of straw from tank
7. Liquid nitrogen tank
8. Microscope

  • Most frozen semen is packaged in .5 ml PVC plastic straws sealed on each end.
  • Semen is removed from the storage tank with forceps and placed in the water bath. It is thawed at a specific temperature and for a specified time as spelled out in the instructions accompanying the semen. BEWARE; rarely a straw can explode when it enters the water bath, sending plastic shrapnel through the air.
  • Complete the insemination procedure by opening straw and depositing semen in syringe barrel.
  • Post-breeding reactions are common due to the lack of seminal plasma in the semen sample.

Seminal plasma is removed prior to freezing and the sperm are re suspended in extender. Seminal plasma is a natural immunosuppressant; it limits the mare’s response in the uterus to the foreign protein of the spermatozoa. Without the seminal plasma the uterus reacts with massive amounts of fluid, protein and WBC production to help eliminate the foreign protein of the spermatozoa.

  • These reactions are often severe enough to interfere with fertility if not addressed by the attending veterinarian with uterine clearance techniques such as lavage and oxytocin injection with or without PGF injections.

FUTURE OF FROZEN SEMEN Selection of good freezing stallions will result in slowly increasing pregnancy rates.

Improved technique and personnel will result in increased pregnancy rates.

Don’t Skip the Purchase Exam
Intracytoplasmic Sperm Injection (ICSI)

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