Artificial insemination (AI) using chilled semen involves the collection, evaluation and dilution of semen from the stallion and then, the TIMELY infusion of an adequate number of sperm into the mare's uterus. The semen must be processed adequately to insure that it retains its fertilizing potential. 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 and 6) Reduces the risk of venereal diseases spreading throughout a breeding population.

When facilities are available cooled transported or chilled semen, provides additional benefits such as the low cost of shipping a container and the dramatic increase of the gene pool. Semen from most stallions can be shipped and delivered to the mare's home within 36-48 hrs after collection. This dramatically increases the gene pool selection for mare owners.

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 cooling and/or the freezing and thawing process. Therefore stallion variability is an added problem.

For an AI programme to be successful strict attention should be paid to health precautions and hygiene. Guidelines and Codes of Conduct to reduce the risk of disease transmission should be strictly adhered to.

Semen collection and handling
All breeding stallions should have a semen evaluation done prior to the breeding season. Most stallions can be easily collected artificially regardless of their age or their previous breeding method. Since most stallions only breed a very limited number of mares semen evaluations are not cost effective. However stallions that will be used in artificial insemination programs regardless of their book size, should be evaluated. Reproductive management of the stallion can have a tremendous positive or negative impact on the stallion's success for that season. This becomes even more important with stallions having marginal semen quality.

Semen can be collected with one of several models of artificial vaginas and the Hannover model is illustrated (Figure One). Although shape and size vary, AV's work with the same principle. A double rubber liner with a water jacket in between. The stallion will ejaculate in response to appropriate temperature, pressure and friction of a well lubricated AV. As a general rule the inner liner temperature of the AV should be between 43°C and 45°C. Selection of temperature and pressure will depend on stallion's preference. The AV should be well lubricated with either vaseline or a sterile nonspermicidal lubricant. The receptacle where the semen is collected should be dry, clean and isothermic in order to prevent cold shock of the sperm cells. Stallions can be collected either mounting a mare in heat, a phantom (Figure Two) or on the ground with proper manual stimulation.

Semen should be collected into a pre-warmed container, and should be maintained at body temperature while in its raw state. Semen that is collected for artificial insemination purposes should be filtered to remove the gel fraction (Figure Three) and diluted with an appropriate pre- warmed extender. The physical characteristics of the ejaculate, including volume, concentration, colour and motility should be evaluated (Figure Four).

Semen preservation
Temperature at which semen should be stored depends on the period of time for which should remain viable. Semen which is to be inseminated immediately can be used undiluted, but if the semen is to be used after 10 minutes it is best diluted with extender. Semen that is going to be used within 6 to 12 hours after collection can be stored at room temperature in a dark environment. Removing the seminal plasma by centrifugation, thereby delaying the process of sperm capacitation, may be beneficial for certain stallions. It is preferable that semen stored for such a period should not be subjected to fluctuations in temperature and storage in a thermos flask should be considered. The semen should be kept at room temperature (18-22°C) in a light-proof, air-tight container.

If semen is intended to last longer than 12 hrs, either because of stallion unavailability or need for semen transport, it should be cooled down to 5-8°C over a 2-3 hrs period to prevent too much energy loss of the spermatozoa. The gel-free ejaculate is diluted with a suitable extender at 37°C. Motility is checked using a microscope with a warming table at 37°C. Each shipment of fresh or chilled semen has to be accompanied by documents with information on the stallion, the collection centre, collection date, shipment date and information about the semen quality and the number of sperm sent. If life expectancy of the semen is longer than 72 hrs cryopreservation (freezing) should be seriously considered. Semen that is collected should be diluted with an appropriate extender regardless of it's intended use. It is extremely important to insure that the extender that is going to be in contact with the sperm is pre-warmed. Failure to do so, results in cold shock to the sperm. Cold shock will result in membrane and acrosomal changes resulting in premature cell death and/or abnormal motility patterns. The cellular changes resulting from cold shock in most cases will severely compromise the fertilization potential of spermatozoa.

The rate of cooling from room temperature is critical and approximately 12 years ago, the Hamilton-Thorne company of Massachusetts developed a container (The Equitainer©) for transporting equine semen. This consists of a strong container with a snap-lock containing frozen canisters, insulation, thermal ballst bags and an isothermaliser. The container is special in that it has the ability to lower the temperature of the semen package approximately 0.3°C every minute until the temperature stabilizes at 4°C to 6°C. The semen should remain constant at that temperature for almost three days. This cooling rate was designed specifically after trials were conducted to determine sperm motility after cooling at various rates. The cooling rate is controlled by a combination of frozen packs, a special copper plate between the packs and the semen, and 60ml of thermal ballast which surrounds the semen. If the total volume of the semen and thermal ballast lies between 120ml and 170ml, the semen will cool at the proper rate.

The author has described the Equitainer in detail to make readers aware that special equipment is required for optimal survival of semen after transport. The Equitainer is easy to use, strong and works very well. Many Equitainer "clones" are available on the market, some at considerably less cost than the original and although acceptable results are often obtained with these clones, sperm survival is probably not optimal. In the authors opinion, until data is available which demonstrates the efficiency of competing units, breeders in the UK should be encouraged to use the original Equitainer.

In the Netherlands a polystyrene box in which two 15ml tubes with chilled semen can be placed is used to transport chilled semen. The polystyrene box is packed in a cardboard box which can be placed in a refrigerated (at 5°C) transporter. This is an efficient and inexpensive system.

Some stallions have semen that transports well in the cooled state, while others do not. For that reason, it is advisable to test the storage ability of a stallion before his semen is shipped to a customer. An ejaculate should be collected, diluted 1:2 with an extender such as heated-cooled skim milk, placed in an Equitainer, then examined at 12 hour intervals for sperm motility. Because one usually packs at least 500 million progressively motile sperm per insemination dose, dilution rates will vary (1:1 to 1:6) according to the density of the semen collected.

When one receives shipped, cooled semen, it is not necessary to warm it to body temperature before insemination. It is important that any transported semen is accompanied by paperwork that advises that suitable health precautions have been taken. It is highly recommended that all the semen in the Equitainer should be inseminated as soon as the shipment arrives. Although many breeding farms ship semen for two inseminations 12 hours apart, part of the semen should not be stored in the Equitainer and used 12 to 24 hours later, in case ovulation has not occurred. The oviduct (falllopian tube) of the mare is a far better incubator of spermatozoa than any transport system available. In fact, it is not uncommon for mares to become pregnant when ovulations occur four, five, six or even seven days after insemination. This is far less likely with stored semen, even when an Equitainer is used because most sperm are immotile (and probably dead or infertile) by 60 hours after collection. Somewhere below 20°C, acrosomal changes occur in the spermatazoa and it is known that semen stored at 4°C has significantly shortened longevity. It is therefore recommended that insemination with chilled semen occurs not more than 24 hours before ovulation. That is not to say that pregnancies will not occur when the interval exceeds 24 hours, but differences between individual stallions, different extenders and different systems of cooling may cause a wide variation in the longevity of chilled semen not only in storage but also in the mare's reproductive tract.

Timing of insemination
In many cases, mares which are intended for AI are often kept at the owner's home where no teaser stallion is available for detection of oestrus. This is likely to be true if, as is often the case for ease of examination, the mare is kept at the veterinarian's premises. Detection of oestrous behaviour in the absence of a teaser stallion is usually misleading and, therefore, the attending veterinarian must be prepared to induce and diagnose oestrus in the absence of a stallion. By a combination of daily rectal and vaginal palpation and ultrasound examination an experienced veterinarian can usually make an accurate prediction of when ovulation will happen.

It must be realised that this involves considerable veterinary costs with several visits and gynaecological examinations necessary to pinpoint the time of insemination. On average, veterinary costs can be approaching £100 per cycle and this will only cover the insemination of the mare. Remember that only about 60% of mares will go in foal on the first cycle. Livery costs and subsequent pregnancy scanning costs will be incurred. These costs are all in addition to the fee for collection and shipping of the semen. Typical costs for collecting semen are £50 with £10 for semen extender; to this are added transport costs for the delivery of the semen and return of the Equitainer. This it is clear that AI is NOT a cheap alternative to natural breeding.

During the ovulatory season, the mare shows repeated oestrous cycles of approximately 22 days in length; 3 to 7 days of oestrus; followed by 15 to 17 days of dioestrus when she has an active progesterone-secreting CL in her ovaries and she rejects the stallion. Following on from this, about one third of mares at any one time in the breeding season will be either in oestrus or coming into oestrus when first examined. Prediction of ovulation is not easy and involves taking into consideration several findings and making a considered judgement. Accurate prediction of ovulation is important because the optimal time for AI with chilled semen is in the 24 hours leading up to ovulation.

Obviously only mares with no signs of uterine inflammation (endometritis) should be inseminated. In young mares with no uterine fluid accumulation and normal pattern of uterine oedema, uterine culture and cytology may not be necessary. However in mares with intrauterine or cervical fluid, culture and cytology should be performed from the uterus. (If the mare has a uterine infection antibiotic therapy should be started). ALL mares that have been barren for more than one year should have a uterine culture, cytology and in most cases a uterine biopsy.

During oestrus, your veterinarian should continue to monitor the cycle by daily 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

Ideally the stallion owner should be notified 48 hours prior to the desired breeding date and a clear communication channel opened between the mare owner's veterinarian and the stallion owner early in the cycle. Any application for import permits should have been made well in advance of the desired breeding date.

Hormonal treatments used to induce ovulation during estrus: Since the hardest part of AI in horses is determining the optimal time to inseminate a mare, hormones are frequently used to induce ovulation. The most commonly used method is the intravenous administration of 3000 iu human chorionic gonadotrophin (hCG) once it has been established that the mare is in oestrus with a soft follicle at least 35 mm in size. Approximately 85% of mares will then ovulate in the 24 to 48 hour period following hCG administration.

Insemination Technique
The mare should be identified from a passport or similar identity document and may need to be matched against those supplied with the semen. If semen has been imported into the UK the accompanying documentation should be checked and it confirmed that the stallion is seronegative for equine arteritis virus.

She should be prepared for insemination in a clean, well lit environment; stocks for restraint are preferable. Her tail should be bandaged and tied out of the perineal region. The vulva and perineal area should be thoroughly cleansed with dilute povidone-iodine solution or mild soap. this is then thoroughly rinsed off with fresh warm water and the perineal area dried with clean, soft disposable (paper) towels.

If chilled semen is being used the semen container should remain unopened until this stage. No attempt should be made to warm the semen prior to insemination of the mare. Since there may be a small delay between cleaning the mare and insemination, it may be helpful to empty the mare's rectum of faeces to prevent contamination of the area after cleansing.

The semen should be gently mixed prior to loading into a sterile plastic syringe without a rubber plunger. The syringe should then be attached to a sterile insemination pipette. The operator should use a 'sterile' obstetric glove (i.e. a glove turned inside out). In cetain circumstances a sterile surgeon's glove should be placed over the clean rectal glove. It may be necessary to place a small amount of sterile, non-spermaticidal lubricant (e.g. KY Jelly) on the top of the hand around the knuckles. The catheter should be held with the tip behind the finger tip and the hand brought into the vulva. The external os of the cervix should be located with the index finger and a finger inserted into the cervical canal. The catheter is inserted alongside the finger and the catheter gently pushed forward. It is very important that the catheter reaches the mid or cranial uterine body and does not remain obstructed in the cervix. This passage through the cervix is not always as easy as may be thought. Deposition immediately cranial to the cervix should also be avoided. The syringe should be gently emptied, infusing the semen into the uterus. During the deposition of the semen it is important that the tip of the catheter is not buried in the uterine mucosa or a uterine fold and any resistance to the flow of semen should be corrected by a fractional withdrawal of the catheter. There is general agreement that within the range of 0.5ml to 60ml the volume of the inseminate is not important. A small amount of semen should then be warmed to 37°C after which it should be examined for progressive motility and gross abnormalities.

Examination following Insemination
The mare should be checked for ovulation by your veterinary surgeon within 24 hours. It may be necessary to order a second delivery of semen if the time of ovulation has been miscalculated.

Post-Insemination Treatment
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
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. The perineal conformation of the mare should also be checked and a Caslick operation performed if necessary.

Insemination dose
The insemination dose required to impregnate a mare is the number of sperm deposited in the uterus at the time of breeding and is not to be confused with the volume of the insemination dose, which is the amount of extender that the sperm are in suspension.

Mares bred artificially are normally inseminated with 100-500 million progressively motile sperm (pms). However it has been accepted as an industry standard the use of 500 million pms to maximize pregnancy rates with fresh semen. Semen collected for chilling and transportation be packaged containing at 1-2 billion pms to allow for some deterioration of the sperm during transport.

The volume of extender use to suspend the sperm cell in an insemination dose appears to have little effect on the pregnancy rates. Doses can vary between 0.5ml up to 100ml with no significant effect on fertility. A typical insemination volume for fresh semen rages between 10-30ml and 30-60ml for fresh chilled semen. Volumes above 100ml should not be used to inseminate mares.

Diagnosis of early pregnancy using ultrasound
The 14 day pregnancy is 13mm to 18mm in size and lies centrally in the uterine body. The embryonic vesicle grows at a rate of approximately 3.5mm/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
The success of breeding with transported semen depends on several factors: 1) Stallion; 2) Semen handling and packaging, 3) Management of the mare and timing of insemination and 4) Mare status. The success for transported semen, under ideal circumstances, is comparable with that of fresh semen. However if semen is not used within the proper timing, success drops to around 20-30% per cycle.

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.

Costs using chilled semen
Using chilled semen is an expensive business. Costs include collection of the semen and transport costs as well as 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 chilled semen:

The first insemination will be £175 + VAT. This will include all examinations, swabs, drug treatments and the insemination as well as all keep and handling fees should be arranged. 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 chilled semen by Equine Reproductive Services will be kept at Busk Hill Stud (Owner: Mr Max Abrams); Westow, York, North Yorkshire YO6 7LS (Tel: 01653-658 288).

If the mare fails to become pregnant subsequent inseminations will be performed at an all-inclusive cost of £150 + VAT per cycle.