Introduction

Artificial insemination (AI) with frozen semen involves the TIMELY infusion of an adequate number of sperm into the mare's uterus. To ensure success in an AI programme knowledge of the reproductive physiology of the mare is very important. Mare selection as well as close monitoring of the cycle are key factors that will determine the success of a breeding programme

A successful AI programme depends upon:

STALLION

• a thorough examination for breeding soundness on the stallion being used
• confirmation that the stallion has semen of sufficient quality
• appropriate cooling and storage of the semen sample after collection

MARE

• a satisfactory breeding soundness examination
• the induction of an ovulatory oestrus
• the accurate prediction of ovulation
• correct timing of insemination relative to ovulation
• appropriate storage, thawing and handling of semen
• correct insemination technique
• post-insemination examination and treatments as required
• correct pregnancy diagnosis 14 to 16 days after insemination

It is important to ensure that the owner is aware that A.I. in the horse requires a high degree of veterinary input. At the initial contact with the mare owner it should be made clear that A.I. is not a cheap alternative to natural breeding. It is vital that there is good communication at all times between the mare and stallion owner and the respective veterinarians.

ADVANTAGES AND DISADVANTAGES OF AI

Although AI has many advantages, there are some drawbacks that veterinarians as well as farm managers must consider before hand.

Advantages of using AI include:

1. Maximize efficiency of stallion usage
2. Evaluate the semen on a regular basis
3. Use of extenders with proper antibiotics to preserve the longevity of sperm and minimize bacterial contamination
4. Implementation of minimal contamination breeding techniques for mares that are susceptible to endometrial infections
5. Enhances the safety of animals and animal handlers.

With the increasing acceptance of frozen semen by the equine industry, stallion and mare owners should be aware that shipping frozen semen is easy and results in the lowest cost per breeding provided that several insemination doses are shipped at once. Frozen semen further increases the gene pool since virtually any stallion can potentially be available any place in the world. By collecting and freezing the stallion's semen, owners will circumvent the conflicts of breeding and competition. Frozen semen also provides a better timing of insemination since in most instances the semen will be shipped several weeks or days before the mare needs to be bred. From the stallion owners perspective collecting and freezing a bank of semen may serve as insurance in case of accident or untimely death of the stallion.

The higher costs due to labour, paper work, the requirement of adequate infrastructure the risk of genetic, viral or bacterial disease transmission are among the disadvantages of artificial insemination. Furthermore semen from some stallions will not tolerate the freezing and thawing process. Therefore stallion variability is an added problem.

SEMEN PRESERVATION: LONG TERM PRESERVATION

Long term preservation refers to semen that will be used more than a few days after collection. The only successful way to preserve sperm for long periods of time, i.e. months or years, is cryopreservation or freezing. Different stallions tolerate differently the freezing and thawing process, and unfortunately the number of motile sperm after thawing is a notoriously poor indicator of fertility of frozen-thawed semen.

In general the freezing process involves the collection of the stallion, evaluation of the semen, dilution and centrifugation, resuspension of the sperm in freezing extender. Unfortunately frozen- thawed sperm appear to have a shorter life than fresh sperm. Due to the apparent short lifespan of frozen semen timing of insemination appears to be critical when using frozen semen.

Thawing and Evaluation

TECHNIQUE

Placement of the semen in the correct location is imperative to achieve good results when breeding artificially. The following steps should be followed to ensure maximal reproductive efficiency in artificial insemination programmes using frozen semen:

1. Make sure that the mare is in heat and is free from uterine infection/inflammation. Rectal palpation and ultrasonographic examination of the uterus should be performed. In mares with intrauterine fluid a culture and cytology should be performed from the uterus. If the mare has a uterine infection antibiotic therapy should be started. Frozen semen should normally only be used in fertile mares.
2. Bring the mare in heat and continue to monitor the cycle by every other day rectal & ultrasound and/or teasing if available. Ultrasonographic examinations should be done prior to breeding. These exams provide information as to the number of follicles of ovulatory size, prevalence of uterine cysts and any sign of inflammation and/or infection. The oestrous period is monitored by daily rectal and ultrasound exams during the first 2-3 days.
3. Mares on the 4th or 5th day of oestrus are given a 3000 IU injection of hCG and then examined at least 12 hour intervals. Mares should be bred very close to ovulation (less than 12 hrs) prior or maximum 4 to 8 hrs after ovulation. The use of frozen semen therefore requires a labour intensive mare management.
4. Every mare should be checked for ovulation and make sure that there is no fluid accumulation in the uterus. If she does have fluid, then a uterine lavage with 1 litre of saline should be performed and oxytocin injections given. Mares that have not ovulated after the desired period of time should be inseminated again.

It cannot be assumed that providing sufficient numbers of viable sperm have been inseminated at the optimum time relative to ovulation that pregnancy will automatically ensue in a mare.

Inflammatory response and Post-Insemination Treatment

Breeding induces an acute inflammatory response which is normal and beneficial. The reason many mares, particularly old mares fail to become pregnant is defective uterine clearance of this inflammatory exudate. It is the spermatozoa themselves that elicit the most acute inflammatory response. A detailed discussion of the inflammatory response to breeding and the management of the susceptible mare is beyond the scope of this paper and only brief details are given here.

Uterine fluid

Ultrasonic examination of the uterus 12 to 24 hours after insemination often shows collections of fluid. these must be removed if optimum pregnancy rates are to be achieved. Oxytocin is probably the drug of choice. Subsequent intrauterine antibiotic may also be beneficial in certain cases. Recently it has become apparent that mares with markedly defective uterine clearance are better treated in relation to insemination rather than waiting for ovulation. Large-volume lavage with warm saline solution in addition to oxytocin may be beneficial in these mares.

    5. Perform a Caslick's operation if the perineal conformation of the mare is poor.
    6. Perform a 14-16 day ultrasound pregnancy examination.

DIAGNOSIS OF EARLY PREGNANCY USING ULTRASOUND

The 14 day pregnancy is 13 to 18 mm in size and lies centrally in the uterine body. The embryonic vesicle grows at a rate of approximately 3.5 mm/day at this early stage of pregnancy and remains highly mobile, making thorough examination of all parts of the uterus important. In the event of twin pregnancies, both vesicles can usually be seen at 14 days, even if the second co-twin arose from a later ovulation. This fact, together with the mobility and relatively small size of the concepti make 14 to 15 days the optimal stage of pregnancy to diagnose twins and crush one co-twin.

Although pregnancy diagnosis is highly accurate even at this early stage, it is important to be aware of the possible confusion caused by uterine cysts and the presence of twin conceptuses. Ideally one would have performed an ultrasound examination before breeding the mare, but this is not always possible. If the first scan is performed at Day 14 or 15, then it is possible to return the next day in cases of confusion and see if the pregnancy has changed position or grown in size. This should allow differentiation from a cyst before the pregnancies have a chance to become unilaterally fixed. Following an initial examination at Day 15, the aim of the examination at Day 24 to 27 should be to assess the embryo is developing normally and identify the heartbeat. It can be confirmed that there is only a single conceptus. If twins were missed at the earlier examination, it may still be possible to correctly manage them.

Ideally a third examination should be performed around Day 33 to 35 to confirm that a single conceptus is developing normally. If there is failure of normal development or if twins are detected, it is usually possible to terminate the pregnancy and re-breed the mare.

PREGNANCY RATES WITH FROZEN SEMEN

The success of breeding with frozen semen depends on several factors: 1) Stallion; 2) Semen handling and packaging, 3) and 4) Mare status. For any breeding operation it is important to realize that the end product (foal) is an interaction between three major factors: Fertility of the stallion, fertility of the mare and managerial practices. Because these factors have a multiplicative effect they should be maximized to ensure maximal reproductive performance for the operation. The biggest factors that affect pregnancy rates with frozen semen are: 1) Stallion, 2) Semen processing and handling, 3) Management of the mare and timing of insemination, 4) Mare status and 5) Experience of inseminator. The success for frozen semen, under ideal circumstances, is almost comparable with that of fresh semen. However if semen is not used within the proper timing, success drops to around 20-30% per cycle.

On the average fertility rates of frozen semen are around 50% but vary between 30 and 65%. First cycle pregnancy rates for foaling, maiden and barren mares are 44, 62.7 and 43% respectively. However the season pregnancy rates (after 3 cycles) are around 80-85% ranging between 60-100%. On the average 2.5 cycles are required to achieve maximal conception rates with frozen semen. When the fertility is analyzed by stallion it is obvious that the range in fertility is even wider. Season pregnancy rates vary between 0 and 100% between stallions and first cycle pregnancy rates range between 0 and 75%. It is recommended that mare owners or individuals using frozen semen inquire regarding the stallion's "first cycle pregnancy rate with frozen semen".

COSTS WITH FROZEN SEMEN

Using frozen semen is an expensive business. Costs include storage of the semen and veterinary and keep costs whilst the mare is being monitored during oestrus. In order to provide owners with an accurate idea of the costs involved, EQUINE REPRODUCTIVE SERVICES Offers a package for mares being inseminated with frozen semen:

The first insemination will be £350 + VAT. This will include all examinations, drug treatments, the insemination and all keep, handling and laboratory fees. Not covered are pregnancy scanning and any Regumate treatment required.

This charge is based on the mare being present at the Stud Farm where the inseminations are being performed for one week. During that time, the mare will be stabled. The keep for mares remaining more than one week at Busk Hill will be subject to the usual livery costs (approximately £7-50 per day, but this should be arranged directly with the owner of the stud farm).

All mares for frozen semen by Equine Reproductive Services will be kept at Busk Hill Stud (Owner: Mr Max Abrams); Westow, York, North Yorkshire YO6 7LS (Tel: 01653-658288).

If the mare fails to become pregnant subsequent inseminations will be performed at an all-inclusive cost of £250 + VAT per cycle.