Fertility, Infertility in the Stallion and AI

 

Reading: Topics 21-27 in Pycock.

        The stallion’s contribution to the success of the breeding enterprise is obviously significant. However, the stallion is often erroneously assumed to have normal fertility. Fertility problems are not uncommon in stallions. However, they frequently go unrecognised. It is often only in retrospect when reviewing the stallion’s fertility records that it becomes apparent that there has been a problem. It is important to know what the expectations of normal fertility are.

          On well managed farms, conception rates of 80-90% are not exceptional. On farms that service a higher proportion of walk-in mares, this may decrease depending on the level of management and veterinary input. Examples of the latter would include Sport Horse stallions, which may have large books of mares submitted on a walk-in basis and without the benefit of veterinary examination to stage the oestrous cycle more precisely. Fashionable and expensive Thoroughbred stallions, well managed, expect to have recorded fertility in excess of 80%.  Many less fashionable and less expensive stallions, who may be forced to accept mares of poor fertility potential, may have recorded fertility percentages lower than this without implying stallion abnormality.  A relatively short and unphysiological breeding season contributes to the comparatively poor fertility figures for Thoroughbred stallions.

 During the investigation of a case of infertility or subfertility in a stallion, it is important that a methodological examination is carried out. Some of the important components of the Breeding Soundness Examination of the Stallion are described below. There are some additional useful reading materials available from the University of Kentucky and these are highlighted on the Web site (www.ul.ie/~equines).

 

1. History

A detailed account of the stallion's past health and sexual history should be recorded.  If the stallion has been standing at stud and is suspected of subfertility or infertility then the teasing, examination, covering and pregnancy records of the mares concerned should be examined carefully.  Poor management and/or a book of ‘problem’ mares can give any normal stallion a low conception or foaling rate.  Study of the mares' bacteriological examination records can give useful information with respect to venereal disease.

 

2. Physical examination

Age, general bodily condition and conformation, such as hooves and limbs are all factors that might contribute to decreased fertility.  The heart is usually examined. 

The reproductive organs should be examined carefully and systematically.  The sheath, scrotum and testes should be palpated with particular regard to size and constituency of the testes and epididymes.  Testicular and scrotal dimensions may be measured with calipers.

Each gram of normal testicular tissue produces 20 X 106 sperm daily and, thus, from testicular size one can predict daily sperm production.

The best time to palpate the testes is after ejaculation, when the scrotum is relaxed, the area seems less sensitive and the testes are less apt to be reflexly withdrawn.  At this time it should be possible to slip the testes and epididymis up and down within the cavity of the tunica vaginalis (the sock like-lining in which each testis is contained). If this is not possible, adhesions are probably present.  Any evidence of trauma to the scrotum should be carefully noted.  The spermatic cord and associated structures may be felt up to the external inguinal ring.  A rectal examination may allow palpation of the accessory glands for signs of obvious abnormality, if the stallion is cooperative.

On presentation with an in-oestrus mare, the penis may be further examined for abnormalities or signs of trauma.  With an excitable stallion, this procedure is best carried out with the mare on the opposite side of a teasing board.

 

3. Bacteriological examinations

Swabs, previously moistened with transport medium or sterile distilled water, should be carefully taken from the urethra, urethral fossa and prepuce. 

 

   The presence of organisms which have been associated with venereal disease (Taylorella equigenitalis, Klebsiella, and Pseudomonas aeruginosa) must be distinguished from the large number of bacterial and fungal surface contaminants often isolated from these sites in normal healthy stallions.

 

4. Sexual behaviour

The stallion should be watched teasing and covering an in-oestrus mare.  Libido may be estimated when the stallion is presented with the mare and his behaviour pattern observed.  The stallion's ability to enter and ejaculate can also be observed.  The number of mounts and time from intromission (insertion of the penis into the vagina) to ejaculation should be recorded, and any antagonistic behaviour noted.

 

5. Semen collection

Semen may be collected by several methods: 

The examination of dismount samples (semen drips) into a sterile jar is the easiest method but results are unreliable.  The quality is very variable and samples often contain accessory fluid and urine.  The presence of leukocytes (white blood cells), clots or potentially pathogenic bacteria may be significant, but it must be determined whether these materials come from the stallion or from the mare.

Semen may be recovered from the anterior vagina of the mare immediately after service.  This method is inadequate because the assessment of seminal volume is impossible and after the specimen has been mixed with vaginal secretions, its pH, sperm motility, morphology and bacterial content are altered.

Semen may be collected in a condom.  Sometimes technical difficulties are encountered with this method; the condom may burst or come off the penis during mating or dismounting and if this happens part or the whole of the semen sample may be lost.

The best way to collect semen is with an artificial vagina. Various models of artificial vagina are available. Most stallions can be persuaded to thrust and ejaculate into an artificial vagina and a satisfactory sample may be obtained.

·      It is considered sensible for the semen collector to wear a crash hat and strong boots with toe protectors.  

·      The in-oestrus mare should be one that can be relied upon to stand rigidly; she is prepared and restrained as for natural cover.

The water jacket in the artificial vagina should be filled with water at a temperature of 45-50'C. If the internal temperature of the artificial vagina exceeds 50'C there is a strong possibility of damaging the semen.  Approximately three-quarters of the length of the liner should be lubricated with sterile obstetrical lubricant; excessive quantities are likely to contaminate the semen.

·The operator stands on the right side of the stallion and mare, near the mare's hind-quarters.  When the stallion mounts, an attendant on the left side pulls the mare's tail to her left and the semen collector deflects the stallion's penis gently to the right side, guiding it towards the opening of the artificial vagina

· The stallion should be allowed to gain intromission, rather than have the artificial vagina pushed onto the penis.

· Ejaculation may be monitored with a finger lightly held under the urethra, palpating the seminal jets and observing 'tail flagging'.

The collection bulb, bottle or bag should be pre-warmed and firmly insulated, especially in cold weather.  After ejaculation the collection vessels should be carefully removed from the artificial vagina and the semen should be examined immediately.

The gel fraction of the semen is a nuisance during both semen evaluation and artificial insemination. It is usually discarded immediately by filtration through a sterile gauze in a pre-warmed sterile funnel.

 

SEMEN

Stallion semen is a milky white to grey white gelatinous liquid.  The liquid comprises spermatozoa and seminal plasma.

In the testis, the germinal cells or primary spermatogonia develop through the spermatocyte stage to spermatids and then to spermatozoa. The daily sperm production of the average mature stallion is 8.0 X 109 sperm. 

 

Sexual maturity in the stallion, in terms of daily sperm production and                     testicular size, occurs by five years of age.  Sperm production per gram of testis, with age, is constant and thus as testicular size increases, sperm production increases.

 

The extragonadal (in the epididymis and collecting ducts) sperm reserves of stallions, ejaculating daily, is 58.6 x 109 sperm of which 88% were located in the epididymis.  Frequency of ejaculation obviously has a marked effect on the size of the extragonadal sperm reserves.

 

Sperm Morphology

The heads are elliptical, with a length of 6.62 ± 0.22 um, width 3.62 ± 0.19 um. 

The mid-piece is on average 9.83 ± 0.33 um long. 

A variable number of morphologically abnormal sperm are found in the semen from normal fertile stallions.  These abnormalities are described as

1.    primary (failure of spermatogenesis),

2.    secondary (failure of maturation) or

3.    tertiary (damage occurring during or after ejaculation).

Large heads may contain the diploid complement of chromosomes and may have two tails.

Most morphologically abnormal sperm have secondary tail abnormalities, often an L-shaped bend of the tail. Most of these sperm are alive but cannot move progressively.

 

Semen evaluation and prediction of fertility

While there are reference values for ‘normal’ semen constituents in 'normal' fertile stallions, there is great variation from stallion to stallion, and at different times of the year.  This, and our lack of knowledge of the correlation between semen constituent measurements and fertility, makes the job of stallion 'fertility testing' very difficult.  It requires specialized equipment, some degree of expertise and experience and often a great deal of time and patience.

 

Semen examination

The semen sample should be examined as follows.

Gross appearance.  The colour of the semen sample should be milky white, evenly turbid, without clots and there should be no unusual smell. 

Any pink or red colouring may suggest haemospermia (blood in the sperm), which is detrimental to fertility.

Volume.  The volume of the semen sample should be measured in a pre-warmed, sterile measuring cylinder.  The total volume of the ejaculate should be 60-120 ml and the gel-free volume 50-100 ml.  These figures are highly variable between stallions and with season and pre-collection ejaculation frequency.

Motility.  The motility of gel-free semen should be estimated immediately after collection on a pre-warmed microscope slide. This method is highly subjective and very dependent on the experience of the examiner.

Motility is classified, in percentage form, as oscillatory or progressive, i.e. those that are alive but are moving on the spot and those that are actively moving forward.

Sperm from a normal fertile stallion should have an immediate motility of 80% oscillatory and 60% progressive.

 

Longevity.  Semen samples may be examined on warm microscope slides at five-minute intervals up to 30 minutes.  If the semen sample is kept at body temperature, the progressive motility should fall off very little over the 30-minute period, but at room temperature, progressive motility starts to deteriorate after 15 minutes.

pH. A pH of between 7.3 and 7.7 is considered normal.

 

Sperm concentration.  Sperm concentration may be measured using a counting chamber with a microscope (haemocytometer) or using a Spectrophotometer, where the estimate is based on the density of the solution.

The concentration of sperm is unimportant if it exceeds 10 X 106/ml. Concentrations of between 100 and 350 X 106 /ml are common, depending on the seasonal influences and ejaculation frequency, and  usually fall in the range of 50 to 100 X 106 /ml.

One method of assessing a stallion’s fertility is to perform a collection of two semen samples, one hour apart: 

1 .The volume of both ejaculates should be the same.

2.    The total number of sperm in the second ejaculate should be half that of the first.

3 .   The pH should stay the same or increase slightly.

4.    The motility should stay the same or increase.

Live/dead ratio.  Smears are made using an equal volume of semen and (nigrosin eosin) stain.  The heads of live sperm remain unstained while the heads of dead ones are stained. The results are variable but you should see a minimum of 50% live and usually more than 60%.

 

Morphology.  The spermatozoa are examined after nigrosin-eosin stain.  They may also be examined unstained. There is an inverse correlation between percentage sperm with primary abnormalities and the fertility of the stallion.

A high proportion of primary abnormalities suggests a testis defect, whereas a high proportion of secondary abnormalities suggests an epididymal defect.  Changes in seminal plasma may affect sperm morphology and so render them more susceptible to damage from manipulation after ejaculation.  Spermatogenesis and/or the passage of sperm through the epididymis may be affected by local increases in temperature, a generalized rise in body temperature, the action of some drugs and over-conditioning.

 

Normal live sperm per ejaculate. It is generally accepted as an industry standard that 500 X 106 normal live sperm are needed to obtain optimal conception rates with artificial insemination. Acceptable conception rates have been achieved with extended semen using 100 and 300 x 106 normal motile sperm. Total numbers of sperm per ejaculate  vary between 600 and 2000 x 106 depending on seasonal effects and assuming 50% normal live sperm in the ejaculate.  Most stallions are well in excess of these minimal requirements.

 

Cytology (Examination for other cells).  A sample of semen should be stained and examined for the presence of other cell types. Up to 1500 leucocytes per ml may be seen in normal semen samples.  No erythrocytes (red blood cells) should be seen.

Bacteriology. A swab from the semen sample should be placed in  transport medium prior to aerobic and micro-aerophilic culture.

No single test, nor combination of tests, seems to be fully reliable so far. Infertility or subfertility can exist in the presence of apparently normal numbers, motility and morphology of sperm.  It is also true that stallions showing very poor semen quality are capable of producing reasonable conception rates if adequately managed. 

 

These factors are a major 'stumbling block' to our ability to predict stallion fertility accurately.

 

If a stallion is to be considered sufficiently fertile to accept a book of 40 or more mares he is expected to demonstrate normal libido, mating behaviour and ejaculation.  His genital organs should be visibly and palpably normal and should be free from signs of inflammation or evidence to suggest infection or contamination with potentially pathogenic micro-organisms.  He should have the potential for ejaculating 400 x 106 sperm in his first ejaculate, between April and July. 50% should exhibit initial progressive motility, 60% should be morphologically normal, and they should not go below 10% motility in six hours.

Owners are still reluctant to have their stallions evaluated for breeding 'soundness' prior to the breeding season and in some cases this may lead to costly delays in a breeding programme. 

 

INFERTILITY

There have been few well documented statistical reports of infertility in the stallion.  Examinations are seldom performed unless fertility is obviously low, so that stallions with various levels of subfertility tend to go unnoticed.  Many stallions cover too few mares for their reproductive capabilities to be determined accurately Fertility statistics of different stallions are difficult to compare because of the marked influence of managerial influences.

 

Managerial influences

Modern systems of stud farm management have a profound effect on a stallion's sexual behaviour and sometimes on his reported fertility percentage. Modern stallion managers are less selective in accepting their complement of mares, in terms of breeding potential, and mare owners are less willing to cull 'bad breeders' than was previously the custom.

In the wild state stallions cover mares many times during an oestrous period.  They remain in contact with the mares throughout and are able to tease them and cover them at their own discretion.  Under systems of modern stud farm management, mares and stallions are separated except for the act of mating.  Teasing is usually performed by another entire horse, kept specifically for that purpose.

Stud grooms and managers usually rely upon advice from the attending veterinary surgeon to decide on the optimal time for covering.  Valuable stallions are restricted to three to four matings a day and 80-100 mares per breeding season, each seldom being covered more than twice per oestrus.

·      General management factors such as lack of or excessive discipline, the stallion's relationships with the stallion man, housing and nutrition may all affect the ability of the stallion to perform his natural function.

·      Underfeeding the stallion during the breeding season will cause physical deterioration which will adversely affect sexual performance.  Emaciation causes testicular atrophy (reduction in size) and poor semen quality. Overfeeding the stallion during the season will lead to physical deterioration and poor, slow sexual performance.  This is probably a physical and psychological phenomenon rather than a direct effect on semen quality.  Incorrect feeding may lead to problems such as laminitis which can have a serious effect on the stallion's sexual performance.  Vitamin supplements, especially vitamin E, are occasionally fed to stallions to improve sexual performance or correct reduced libido.  Although vitamin E is essential for normal reproductive function in the rat, there is no evidence that additional vitamin E is helpful in the stallion.  There is no reliable evidence that mineral deficiencies have a specific effect on the reproductive tract of the stallion. Provided food intake is adequate and work and exercise correctly balanced, the benefit of feeding supplements is questionable.

·      Stallions are required to mate outside the optimal period of the year for libido and at times of the day when their natural inclination may dictate otherwise.  In these circumstances the number of services per day may be significant and particular individuals may suffer from overwork if called upon to mate three or more times in 12 hours for seven consecutive days and several consecutive weeks.  This may result not only in a loss of libido but also, early in the breeding season, in the depletion of extragonadal sperm reserves and the ejaculation of semen of low sperm density. Such problems might be avoided by artificially increasing daylight length during the winter or early spring to improve semen quality and sexual behaviour.

·      Failure to recognize ejaculation is one of the most serious defects of management in the covering yard and can be a substantial cause of subfertility.  Some stallions show obvious signs of tail-flagging; others are very deceptive.  The only reliable method of diagnosis is to place a hand lightly on the under-surface of the penis after intromission and feel the ejaculatory pulsations in the urethra.

 

Abnormal behaviour

The most common cause of abnormal sexual behaviour is mismanagement.  In many instances, if a young stallion develops behavioural abnormalities such as difficulties in intromission or ejaculation, the remedy is to encourage coitus without restraint of mare or stallion. The disciplinary measures presented to race-horses in training may result in a suppression of normal sexual behaviour when a stallion first retires to stud. A quiet, experienced mare well in oestrus and with an unsutured vulva is a good subject for a first mating.  A traumatic first experience for a young stallion may cause lasting psychological problems.  A young stallion may become confused if too much human guidance is given. The establishment of a good stallion/handler relationship is essential for good breeding routine and adequate sexual performance.  For this reason only one individual should handle the stallion and he/she should be patient and understanding.

There are variations in the number of mounts per ejaculation and reaction time in and out of the natural breeding season.  There is a greater tendency for stallions to show aggressive behaviour towards mares out of the breeding season.  It is difficult to cure a stallion of an aberrant behavioural pattern once established.  Such stallions may become excessively vicious, biting and kicking the mare instead of mounting. 

Systemic illness or pain may result in abnormal sexual behaviour, often manifest as reduced libido or failure of erection, intromission or ejaculation.  Any stallion showing abnormal sexual behaviour should receive a thorough physical examination for systemic illness.  The genital organs should be examined for signs of injury or infection. 

The mature stallion who loses his libido halfway through the stud season may have problems involving management, general health, injury or inheritance.  Failing libido may follow overwork, detrimental handling or adverse changes in the stallion's environment.  A particular mare to which a horse takes a dislike or with which he has had a painful experience may reduce his confidence, especially if management persists in attempts to force a successful mating. 

·A stallion that shows little or no sexual interest may be lunged for half an hour before presentation of the mare.  This, or a change in surroundings at mating time, may increase libido. 

·The stallion may be used to tease mares in the morning and this may accentuate sexual desire in the covering yard.

The habit of prolonged delay in mounting or ejaculating, so that a service takes 30-60 minutes, frustrates horse and management. 

·      Various procedures can be tried to encourage more speedy performance, from the use of hippomanes to injections of testosterone, from the administration of LH, GNRH or women's perfume applied to the mare's hind-quarters.  No remedy appears to have had consistent success.  Perhaps the best advice is to limit the stallion to a maximum number of mounts - say three - before returning him to his box.  A further attempt is then made an hour later.  Stallions who take a long time to cover may have to be restricted to a reduced number of mares during each season.  In certain instances turning the stallion loose with a mare in oestrus may help to restore his confidence and reduce the time he takes to mate.

Many of these cases are primarily psychological and respond to a retraining process. Each case is different and successful treatment requires close observation.  Sometimes such a case may respond to the sight of another stahion covering a mare.

Genetic factors

Sterile or subfertile stallions with no history of trauma, infectious disease or managerial problems are often considered to have genetic inadequacies.  These may be associated with semen abnormalities or, in rarer cases, chromosomal abnormalities.  Hermaphrodites have been reported but are usually associated with obvious abnormalities of the genitalia.  Penile size is probably determined genetically and in some cases may produce mechanical difficulties at covering.  Ideally, horses suffering from heritable conditions should not be used for breeding, as abnormalities may thus be spread among the horse population.  Cryptorchidism (rig) is a heritable condition and may be associated with degrees of testicular hypoplasia in pony stallions.

Umbilical and inguinal hernias may be genetically determined and in many cases are corrected before the horse becomes a stallion.  This produces an ethical problem which is difficult to solve.  If they are not corrected they may cause unwillingness to cover due to pain or discomfort during attempts to mount.

 

Hormonal factors

There are no reports in the literature of the correlation of blood hormone levels with fertility problems and, although endocrinological inadequacies are frequently blamed for infertility or subfertflity in the stallion, hormonal treatment rarely causes lasting improvement.

 

Abnormalities of the penis and prepuce

The penis and prepuce are very vulnerable to traumatic injury.  If kicked when erect, the penis may swell rapidly vascular rupture and haemorrhage.  Immediate first aid for such an injury should include removal of the mare, quietening the stallion and the application of cold water either by hose or with wet towels.  A similar rupture of a superficial penile blood vessel may occur when covering a tightly sutured mare, without prior episiotomy, or when a mare moves suddenly while the stallion is thrusting. 

Tumours of the penis are uncommon.  Papillomas (warts caused by viruses), angiomas (tumours of blood vessels) and melanomas (tumours of melanin secreting cells) have been recorded in or on the sheath and penis of horses. Sarcoids may involve the prepuce, causing severe discomfort.

The herpesvirus associated with coital exanthema causes painful vesicles on the

glans penis.

 

Abnormalities of the scrotum, testis and epididymes

The scrotum is subject to traumatic injury. This may cause inhibition of spermatogenesis by interfering with heat exchange.

Incompetence of the inguinal rings may result in herniation (protrusion through the inguinal canal) of peritoneal fat and small intestine into the scrotum.  Strangulation of the hernial contents is recognized by acute colic and hard tense painful scrotum. Urgent surgical intervention is essential.

 

Orchitis (inflammation of the testes) may be a complication of injury, for instance a kick, or of infection.  The testes become swollen, hot and painful. The stallion may refuse to cover, but if he ejaculates the semen may contain large numbers of leucocytes, sometimes appearing as clots. The condition may become chronic, with testicular fibrosis and poor semen quality, which may have a permanent effect on the stallion's fertility.  Acute orchitis should be treated with systemic antibiotics.

 

Testicular degeneration may be associated with thermal factors following the elevation of body temperature by systemic infection, very high ambient temperatures and possibly conformation factors resulting in an incompetent heat-exchange system.

 

Abnormalities of secondary sexual organs

Seminal vesiculitis is not uncommon in the stallion.  The condition may be diagnosed by rectal palpation.  One or both vesicles are enlarged and sensitive.  The stallion may refuse to cover, or may try to do so but be unable to ejaculate.  If ejaculation does occur, the semen will contain many leucocytes and bacterial culture will reveal pathogenic organisms. 

Apparent blockage of semen in the efferent ducts, somewhere between the testis and penis, has been encountered. Following excessive sexual excitement the stallion produced a semen sample which was of the constituency of thick cream. Following the apparent removal of this 'blockage', semen samples contained live, morphologically normal and motile spermatozoa and fertility returned to normal.  The exact site of blockage in this case was not determined.

 

SPECIFIC GENITAL INFECTIONS

Equine coital exanthema

Coital exanthema is a specific venereal infection caused by a herpesvirus distinct from equid herpesviruses 1 and 11.  The virus affects both stallions and mares, but the manifestations in the stallion are usually more severe. 

The infection is brought into the stud farm by a visiting mare, usually a symptomless carrier.  She then infects the stallion, who infects other mares, before he shows clinical signs and mating is stopped. The lesions often heal rapidly and stallions can normally recommence their duties in 10-14 days after the first signs.  In the stallion, lesions appear in the form of papules, vesicles or pustules on the penis and there may be associated dullness, anorexia and unwillingness to work.

Treatment is by systemic and local antibiotics (to combat secondary bacterial infection) and sexual rest.  All mares covered by the stallion during the previous ten days should be examined, treated if necessary and isolated.

Bacterial infections

Stallions harbour many kinds of potentially pathogenic and non-pathogenic bacteria on their external genitalia, particularly in the prepuce and associated smegma. The stallion does not usually suffer clinical signs and the bacteria are more important for their effect on the fertility of his mares.  Bacteria isolated may be classified according to their effect on the mare:

1.   Those organisms not associated with infection and endometritis in the mare,

2. Organisms capable of causing infection and endometritis in mares who have depressed local natural defence mechanisms (e.g. Streptococcus zooepidemicus (,6-haemolytic streptococci), Staphylococcus aureus (coagulase-positive), haemolytic E. coli.

3.   Organisms capable of causing primary infection and endometritis in mares with normal local defence mechanisms, and outbreaks of venereal disease, e.g. some Klebsiella, some strains of Pseudomonas aeruginosa and T. equigenitalis.

Any of these organisms may be mechanically transmitted directly into the uterus of the mare at coitus.  It is impossible to sterilize the penis and prepuce before service and coitus results in further bacterial challenge to the endometrium showing no clinical signs himself. Similarly, with contagious equine metritis stallions show no systemic signs during an outbreak.

For the detection of possible venereal-disease-producing bacteria on the genital organs of stallions, swabs should be taken from the urethra, urethral fossa, prepuce and pre-ejaculatory fluid on a number of occasions. (See the Code of Practice)

 

ARTIFICIAL LIGHTING

The physiologic breeding season can be successfully manipulated to fit into the operational breeding season by artifi-                        b

cially increasing the photoperiod.

·      Providing 14-16 hours of light stimulus (artificial plus natural) per day is adequate.                   i

·      Because a lighting program requires a minimum of 8-10 weeks for response, mares in the northern hemisphere are exposed to the lighting system by December 1 to establish normal cyclic activity by February 15.                   d                   v

·      Various methods of light administration have been used successfully, the most common of which are:

(1) using a light source that is held steady at 14-16 hours a day throughout the entire stimulation period, or

(2) increasing light by small increments (similar to that which occurs naturally), usually by adding 30 minutes of daily light stimulation at weekly intervals until 14-16 hours of light exposure is achieved (e.g., 1 0 hours December1, 10.5 hours December 8, etc.).                        c

Instead of adding the supplemental light only at the beginning of the natural daylight period, best results would be obtained when the supplemental light is either added to the end of the natural daylight period or split and added to both the beginning and end of the natural daylight period.   i

French researchers have described a technique for providing a 1 -hour pulse of light 18.5 hours after the onset of daylight. This dark-phase light pulse has reportedly resulted in resumption of reproductive cyclicity similar to the more traditional lighting techniques.

The mare be within 7-8 feet of a 200-watt incandescent light bulb in order to provide adequate light exposure, and the stall should have sufficient window space to permit the same exposure during daylight. Minimal light intensifies have not however been adequately determined.   t

   Shadows can prevent the achievement of desired results, so care should be taken to eliminate them. Paddock lighting systems are also successful if light exposure is sufficient in all areas of the paddock.

ARTIFICIAL INSEMINATION

 

Artificial insemination is not a new technology. Indeed, the horse was used in AI studies as early as the 18th century. In the 1930’s, Russian scientists reported on the use of AI in over 30,000 mares. Despite this, recent advances and developments in AI that are used in other species have not been adapted in equine AI. This was, in part, due to industry lack of interest but is also due to some of the inherent difficulties in freezing stallion semen.

The technique for unfrozen semen is simple and easy to perform and is now used routinely in the Standardbred 'and Quarterhorse breeding industries in the United States and increasingly in the Sport Horse industry throughout Europe. Currently in Ireland, only 8% of mares in the Sport Horse industry are bred by AI. The semen is collected by an artificial vagina, with the stallion mounting an in-oestrus mare or phantom. The semen may then be used in one of four ways:

1. The whole ejaculate, without dilution, may be inseminated into a single mare.

2.   Following, removal of the gel fraction, the semen may be diluted with an extender at room temperature, for insemination into one or more mares.  Unless it is to be used immediately (within 30 mins-1 hour of collection), semen must be diluted with an extender. The most comonly used extender is the Kenney extender which contains egg-yolk, skim milk, sugars and antibiotics. The extender also contains glycerol, which acts as a cryoprotectant to protect the sperm during chilling.

Extended semen has a longer life. It can be safely stored at room temperature (18-22 C), in the dark, if it will be used within 6-12 hours of collection.

3.   If semen is to be used 12 hours after collection, it must be cooled to 4-8 C. Chilling must be gradual to avoid damage to the spermatozoa. The semen,

diluted in extender, is usually chilled over a 2-3 hour period. A company in MA, USA developed a container called the Equitainer, that has the ability to steadily drop the temperature of semen 0.3 C each minute until the temperature stabilizes at 4-6C. The semen is usually shipped in this container which can maintain this constant temperature for approximately 3 days. Stallions vary enormously in the susceptibility of their semen to chilling effects. This can be checked beforehand out by chilling the semen and evaluating it at 12 h intervals. Useful tests might include live:dead ratios, and percent motile sperm. .

4.         For long term preservation (>3 days) sperm must be cryopreserved in liquid nitrogen. Sperm are usually preserved in small volume straws (0.5 ml) at -196 C in liquid nitrogen. To achieve the appropriate concentration in a small volume, the sperm are 'washed' by repeated centrifugation and dilution with semen extender containing antibiotics, nutrients and cryoprotectant such as glycerol. The semen is then frozen gradually.  Centrifugation does not appear to damage sperm motility. Much more information is now available on the techniques of freezing stallion's semen, which is significantly more fragile than bull semen.

The mares for insemination are usually prepared in a collection area and are inseminated with the semen into the uterus via plastic sterile pipettes. The insemination volumes vary considerably but 30-60 mls are common ranges.

With good technique, a high fertility percentage can be achieved by the use of an artificial insemination programme.  The exact number of normal motile sperm needed per insemination is not known.  Success with as few as 100 X 106 normal motile sperm has been reported but in the absence of more accurate data, and to give a practical safety margin, 500 X 106 normal motile sperm is recommended. Raw (undiluted) semen is normally inseminated within one hour of collection and so is used within the confines of the stud farm.  The semen is maintained at 30-35C and precautions are taken to prevent 'cold shock'.

For short-term storage (up to three days) of stallions semen, the semen may be diluted immediately with extender, usually in a 2 : 1 proportion.  The extended semen is then cooled slowly to 4'C over one to four hours and held at that temperature until used.

 

Artificial insemination is widely used throughout the world in non-Thoroughbred horses.  Thoroughbred registration authorities refuse to accept progeny produced by artificial insemination. The advantages of AI are as follows:

1.   Disease control.  By providing a system where there need be no direct physical contact between the stallion and mare, the risk of spread of contagious infectious disease, either venereal (equine coital exanthema, Kl. aerogenes, Ps. aeruginosa and Taylorella equigentalis) or non-venereal (equine infectious anaemia, strangles, equid herpesvirus 1 abortion, equine influenza), may be very much reduced or eliminated. Use of semen in extendrs and antibiotics can also be useful to reduce the normal seminal bacterial contaminants, as a prophylatic measure, in mares suffering from an increased susceptibility to uterine infection.

2.       Overcoming physical deformities in mares.  Mares that have tightly stitched vulvas may be inseminated without the need for continually re-opening and reclosing the suture line.  Mares with physical conditions or behavioural problems which make natural service difficult or hazardous may also be inseminated.

3.            Overcoming abnormal or unwanted stallion behaviour.  Stallions with abnormal

sexual behaviour may be retrained to an artificial insemination programme; vicious stallions may be taught to use a phantom, thus eliminating the risk of damage to the mare.

4.   Managerial advantages.  The collection and fractionation of semen with an artificial vagina could significantly reduce the covering load of a stallion.  This might be a great help in avoiding the deterioration of semen quality during busy periods and in reducing the time spent by stud farm personnel in the covering yard..

5.   It is theoretically possible to synchronize oestrus using prostaglandin and HCG treatment, and then to inseminate, resulting in a reduction in the spread of foaling the following year.  Such a system, if widely used, could reduce the length of the breeding season with all the economic advantages that would accrue.  Using freezing techniques, costs might be reduced by transporting semen rather than mares from country to country. 

6.   An artificial insemination system could increase the breeding efficiency of any particular stallion and could, if considered desirable, significantly increase the number of mares he inseminates.  Similarly, using freezing techniques, the breeding life of the stallion could be increased.

7.   An additional advantage is that semen is evaluated regularly in an AI situation.

 

Registration authorities have traditionally been worried by the risk of exploitation of fashionable sires, reduction of the individual value of bloodstock through oversupply and the increased risk of fraud.  There is also a worry that the traditional organization and structure of the breeding industry would change from stud farms to stallion stations.  Many of these worries stem from an ignorance of how equine artificial insemination systems are run and the misconception that a system such as used in dairy cows might be directly applicable to horses.  There is little similarity between either the needs of the two industries or the necessary technical methods for handling the two types of semen.  It is a great mistake to compare these two different systems.

 

There are also disadvantages to AI in the horse.

1.    At present, the costs associated with the procedure are higher. This may be a factor of scale but the management of the mare does have to be more intensive and timing of AI is more critical than timing of natural service. In general, chilled semen should be inseminated no more than 24 hours prior to ovulation. Frozen semen should be inseminated 6-12 hours before insemination. This will require greater veterinary input.

2.    Insemination is not a trivial undertaking for a stallion centre in that a degree of laboratory infrastructure must be provided.

3.    There is still a risk of transmission of disease. Where the semen is shipped across international borders, there is the risk of introducing new infections. A good example is the introduction of Equine viral arteritis into the UK in the early 1990’s.

4.    Overuse of individual sires with undesirable genetic traits could perpetuate genetic defects in the population.

5.    Stallion variation in the ability of their semen to be frozen is an obstacle to widespread use of the technique.

 

GENERAL SEMEN-HANDLING

TECHNIQUES

 

·      Immediately after its collection, semen should be quickly transported to the laboratory, and physical trauma, exposure to light, cold shock, or excessive heat should be minimized.

·      All materials that come in contact with the semen (including the seminal extender) should be pre-warmed to body temperature (37'-39' C).  If an in-line filter was not fitted in the AV when semen was collected, the semen should be poured through a nontoxic filter to remove any gel or extraneous debris.

·      Spermatozoal concentration, volume and coloor of the gell-free semen, and the percentage of progressively motile spermatozoa should be determined and recorded.

Semen should always be mixed with an appropriate extender within a few minutes after collection to maximize the longevity of spermatozoal viability.  An initial semen-to-extender dilution ratio of 1: 1 to 1:2 is generally adequate if semen will not be stored long (i.e., < 1-2 hours) before insemination.

Properly formulated seminal extenders improve spermatozoal survival during the interim between collection and insemination.  The most commonly used equine seminal extenders are milk-based.  Some milk-based extenders are available commercially. 

Addition of appropriate antibiotics to seminal extenders will aid elimination of bacteria, which invariably contaminate the semen sample during its collection.

 

INSEMINATION TIMING

AND FREQUENCY

 

In many AI programs, mares were traditionally inseminated every other day, beginning the second or third day of estrus, until ovulation was detected or until the mare no longer exhibited signs of behavioral oestrus. This is probably a luxury now and stallion owners are less likely to be this generous. 

 

In a recent study a single-cycle pregnancy rate of 75% (9 of 12 mares) was obtained from single inseminations performed 3 days before ovulation; however, the highest pregnancy rate (88%.. 7 of 8 mares) in that study was in a group of mares inseminated 1 day before ovulation.  The pregnancy rates in mares inseminated within 6 hours of ovulation was comparable to that achieved with a single insemination before ovulation, or two inseminations, one before and one after ovulation. A high embryonic loss rate (34%) occurred in mares inseminated more than 6 hours after ovulation.  Pregnancy rates were reduced and embryonic loss rates were increased in mares bred 12-24 hours after ovulation.  So the bottom line recommendation for fresh or chilled semen is to inseminate 12-24 hours before ovulation.

Ideally, transrectal ovarian palpation or ultrasonography should be used in a breeding program to more accurately predict ovulation

 

INSEMINATION DOSE

(SPERM NUMBER)

 

Usually, mares in an AI program can be successfully inseminated with 250 million progressively motile spermatozoa.  Insemination of mares with 500 million progressively motile sperm will help ensure that acceptable pregnancy rates are achieved by allowing some margin for error in semen evaluation and handling when conditions are less than optimal.  If semen is properly handled and from a highly fertile stallion, the insemination dose can sometimes be reduced to 100 million progressively motile spermatozoa without reducing fertility. One study revealed that mares inseminated with 50 million motile spermatozoa had lower overall pregnancy rate (38%) than did mares inseminated with 500 million motile spermatozoa (75%).

 

 

INSEMINATION VOLUME

·      The number of spermatozoa in an insemination dose appears to be more critical than the volume of the inseminate. 

·      Although smaller or larger volumes can be used successfully, typical volumes for extended equine semen range from 10-60 ml.

·      Timed closely with ovulation, insemination of frozen/thawed semen in volumes as low as 0.5 ml has resulted in pregnancies.

·      IVs greater than 100 ml are not advantageous because much of this volume may be lost through the mare's dilated cervix after insemination.  Often only a small number of mares are to be inseminated with an ejaculate, so it is commonly diluted with extender with the total volume being equally divided among the mares to be bred.

 

INSEMINATION PROCEDURE

·Sterile nontoxic disposable equipment is recommended for AI procedures. 

·Syringes with nonspermicidal, plastic plungers are preferable for AI because rubber plungers may possess spermicidal properties.  Individual stallion variation seems to exist regarding spermatozoal sensitivity to the toxic effects of syringes with rubber plunger tips.  Toxic effects are apparent in semen from some stallions with as little as 1 minute of contact with syringe plungers.

·Insemination of the mare should be performed in accordance with minimum contamination techniques.

· The mare should be adequately restrained with her tail wrapped and diverted either to the side or over her rump.

·The perineal area, particularly the vulva, should be thoroughly scrubbed and rinsed.

·Any dirt or fecal material within the caudal vestibule should be removed during the washing process to prevent contamination of the upper reproductive tract during insemination.

·To inseminate a mare, a sterile shoulder length plastic sleeve is first placed over the arm used for insemination.  The tip of a 20- to 22-inch insemination pipette is then positioned in the cupped hand and a small amount of sterile, nonspermicidal lubricant is applied to the back of the hand. 

·The covered hand and insemination pipette are passed into the cranial vaginal vault where the index finger identifies and penetrates the cervix.  The insemination pipette is then advanced through the cervix to the mid-body of the uterus. 

·A syringe containing extended semen is attached to the insemination pipette and the semen is slowly deposited into the uterine lumen.  Another equally satisfactory method of insemination is to pass the insemination pipette through the cervix using a lighted speculum preplaced in the vagina.

 

SUMMARY

 

AI is an effective technique for improving utilization of stallions in breeding programs.  When proper semen handling and insemination procedures are used, optimal pregnancy rates are attainable.  When AI techniques are used for mares and stallions with marginal fertility, pregnancy rates are sometimes improved over those achieved with natural mating.