Stallion Breeding Soundness Examination
12-15

• The goal of a stallion breeding soundness examination is to select stallions for fertility, eliminate stallions with heritable defects, alert owners of subfertile stallions, and determine any cause of infertility.
• It is important to note that the fertility is assumed for time of examination only, since conditions may arise shortly after the examination that affect fertility.

• The foaling rate is a good indication of fertility.
• Check the foaling rate of the last breeding season.
• Check the reproductive history of the mares, as barren or infertile mares may make stallion look like he has subfertility.
• Record the services/foaling, but be careful as abortions etc. that are not associated with the stallion may alter the number of foals born.
• Calculate the services/conception for maiden, barren, and foaling mares.
• If a problem shows up, you may also want to check the management (breeding and housing) of the mares to help rule out a management problem.
• Determine the intended use of this stallion
  • Natural service vs. AI
  • Fresh cooled semen?
  • Frozen semen?
  • Size of book (expected number of mares to be bred

• The stallion should be free of Equine Infectious Anemia , Equine Viral Arteritis , CEM.

• Positive identification is essential.

• A tattoo is the best identification, but a photo is also a good idea.
• In any case, make sure you positively identify the stallion to avoid legal complications later!

• Conformation,
• Lameness,
• Vision,
• Inherited Defects,
• Cryptorchidism, 2 scrotal testes
• Combined Immunodeficiency,
• Parrot Mouth,
• Hemophilia,
• Complete Mature Cataracts,
• Aniridia,
• Wobbler,
• Multiple Exostosis.
• A breeding sound stallion should be free from these defects.
• Ultrasound
  • Although we usually think of the mare when we consider reproductive ultrasonography, there are a number of uses for ultrasonography in the stallion. 
  • Ultrasonographic examination of the testes is an accurate method for determining testicular size, as well as identifying pathologic features. 
  • Testicular parenchyma can be examined, testicular trauma evaluated and tumors identified. 
  • The central vein is an easily identifiable landmark. 
  • Scrotal contents such as bowel or excessive fluid can be visualized. 
  • Hematocele can be differentiated from hydrocele. 
  • The internal genitalia can also be examined. 
  • The accessory sex glands are better evaluated using ultrasonography than by palpation alone.
     

• Semen is collected with an artificial vagina. 
  • A stallion ejaculates based on the temperature and pressure exerted onto the penis by the vagina, or in our case, the artificial vagina. 
  • Each artificial vagina is designed to produce the correct pressure and temperature to make a stallion ejaculate. 
  • Each model has advantages and disadvantages.

• This is a commonly used model. It forms a water jacket using a hard shell and a rubber liner. A second rubber liner or a disposable liner is inserted into the AV to collect the semen.
• The advantage of this system is that it holds a large volume and will retain its heat very well in cold climates.
• A disadvantage is that it contains a large amount of water and is fairly heavy.
• Another disadvantage is that after ejaculation the semen is retained in the confines of the water liner. If the water is not drained out quickly, the sperm cells are subjected to a deleterious temperature.
• The ARS version has an entire setup (liner, filter, collection bottle) available which includes a disposable liner to prevent contamination of the sample and contamination between stallions. The disposable liner also facilitates clean up. The latex liners are reported to cause lower sperm motility, but some stallions object to the disposable plastic liners.
• The system also includes an in-line semen filter to filter out the gel fraction of the ejaculate.
• If the ARS is completely filled with 50^o C water, it will equilibrate to 45^o C in about 10 minutes.
• The pressure is adjusted by opening the valve, inserting your arm into the AV and letting water out to the desired pressure (unfortunately this is only a guess).
• If more pressure is desired or the temperature needs to be increased, more water must be added (this is a pain when you are out collecting a stallion).
• Dr. Eilt's favorite.............at least for stallion collection.

• The Missouri AV contains the water between two soft liners, instead of a hard shell.
• Advantages of this system include:
• You can easily adjust the pressure by blowing air into the water bladder.
• The semen does not remain in contact with the water after ejaculation.
• It is very light.
• Disadvantages include:
• It does not hold much water, so it drops in temperature relatively rapidly in cold weather.
• It is difficult to get and insert disposable liners into it, so it must be cleaned with alcohol between each stallion. This results in a greater chance of contamination.
• The leather case does not always hold the AV firmly when the stallion's penis enters the AV.
• A special "coupling nut" is needed to attach a collection bottle to it

• Somewhat of a Nishikawa - Colorado hybrid
• Outer case is made of hard rubber
• Has a partially closed distal end for the stallion to push the end of the penis against
• Disposable liners, filters, collection bottles are available, similar to the Colorado
• Dr. Paccamonti's favorite............for collecting stallions.

• This is basically an aluminum Colorado.
• It has a small hole in the cap that is designed to vent water and automatically adjust the pressure as the stallion's penis enters the AV.
• There is also a rubber ring in the end of the AV that is designed for the stallion to push his penis against and supposedly feel more natural.
• It is smaller and made of aluminum, so it loses heat rapidly.
• The collection 'bottle' is a rubber cone, which can be contaminated easily.
• These are no longer available.

Krakow - Polish

• This is just a short model used for .......no not stallions that have a short penis........for fractionating the semen sample.
• It is most useful in situations when you want to examine the ejaculate to determine where a particular problem originates.

See the French IMV by clicking here

• Most stallions want a temperature of 45-48^o C (depends on stallion's preference).
• Adjust the pressure to stallion's preference just before you are ready to collect him. This keeps a lot of water in the AV and helps prevent cooling.
• Use about a tablespoon of non-spermicidal lube when you are adjusting the pressure. Only lubricate abut the first third of the AV. Lubricate just before collection to avoid drying out the lube and to check for foreign objects (thermometers) in the AV. Studies have shown that even non-spermicidal lubricants can have detrimental effects on sperm due to changes in osmolarity. There are some reports coming out about how spermicidal lubes are. These reports tended to use a lot of lube placed into the semen (5% by volume).

• Commercially available
• Hard to put on and keep on
• Have seen human condoms used on mini stallions (best to use two)

Manual stimulation

• This can be used for stallions that have difficulty mounting.
• You can attempt manual massage of the erect penis with moist towels while the stallion is standing or while he is mounting.
• Experimentally it took about 1 1/2 training sessions to train stallions to do this.
• The semen (except for pH) is the same as if done by an AV collection.

• This can also be used for stallions that have difficulty mounting.
• Erection and ejaculation are primarily alpha, whereas arousal beta stimulation.
• Xylazine
• It has both alpha and beta actions.
• Use 0.3 mg/lb after sexual stimulation.
• Experimentally semen was obtained in about 40% of stallions.

• Imiprimine is a tricyclic antidepressant used in humans. (It was noted that a side effect of the drug in humans was ejacualtion)
• It is only used experimentally now.
• The 'current' method used by Sue McDonnell and sent on the Equine List server 3/4/02

  • "We now (2002) use the oral imipramine instead of injectable. (We had trouble getting injectable preparations that didn't result in some worrisome hemolysis.) 

  • For a 1000 pound horse we now get best results starting with 1000 mg imipramine  hydrochloride orally approximately two hours before 200 mg. xylazine IV. Ejaculations when they occur, tend to be at about 2 minutes after xylazine  or at 15-20 minutes after xylazine, either when the horse is just becoming sedate or just regaining alterness.  We adjust the dose for subsequent attempts based on the horse's response.  If he goes from normally alert to fully sedate (head down to about 8 inches from floor) very gradually over 120 seconds after xylazine, then we consider that is a good level of xylazine. If he goes down abruptly (less than 1 minute) or to very deep standing sedation (wobbly swaying, snoring with nose on floor), then too much xylazine.  If he never gets fully sedate (head down to about 6-8 inches from floor, and not alert and responsive to surroundings) or not fully sedate until several minutes after xylazine, then we just that not enough xylazine. These induced ejaculation protocols based on imipramine and xylazine have always worked best with with a quiet stallion that tolerates injections well.  We deliberately do the procedure at quiet times and have a minimum of people to disturb the horse (only one). We attach a collection bag over the prepuce on a girth strap so that after a queit approach and injection, we can tip toe out of the stall and leave the stallion undisturbed. we either monitor on remote video monitor or "peak" around the corner quietly.

  • Also, with this protocol the semen will likely be very concentrated, with a small volume.  That's due to the imipramine enhancing contractions of ampullae and inhibiting somewhat the contractions of accessory sex glands.It's great for freezing, but needs immediate careful handling to avoid cold shock. We've had some over 1 billion per ml.  the total count is usually higher than what you would get in an in copula ejaculate from that horse. 

• Semen collection after sexual rest is not indicative of how many sperm cells a stallion can produce under normal use.
• It is best to collect a stallion when he is at his Daily Sperm Output (DSO). 
  • The DSO estimates the number of cells the testicular parenchyma can produce daily. 
  • This is when all the cells he is ejaculating equals the cells he is producing every day. 
  • This is best done by having the stallion ejaculate every day for 3-5 days. 
  • Normally the sperm output will stabilize at the daily sperm output. 
  • It may take 7-10 days in some cases to get to the DSO. 
  • The mean of last 3 daily collections is DSO. 
  • You can estimate the DSO by taking 27.5% of second ejaculate (taken 1 hr apart). From testicular measurements:
  • DSO=-3.36 X 109 + (0.066 X 109)(SW in mm)
  • DSO=(0.024)(TV)-0.76 .     (TV=(4/3)(heigth X width X length/2).

   

• It would be a good idea to collect a stallion at his normal use schedule.

• For a stallion not trained to a dummy, an estrous mare is needed. 
  • If possible pick one that is calm and does not like to kick. 
  • If you do not have a mare in heat , an anestrus mare given 1 - 2 mg estradiol cypionate (ECP) will show signs of heat. 
  • An ovariectomized (OVX) mare given 2 mg ECP 2-3 days in advance is ideal, in that the temperament for the mare OVXd can be selected, and you 'always' have a mare in 'heat'. 
  • A dummy or phantom is ideal if the stallion is trained for it. Dummies do not move or kick!
• Wrap the mare's tail and tie it off to the side.
• Make sure the restraint is adequate for both mare and stallion.
• Stallion preparation
• Tease the stallion to erection.
• Wash the penis with warm water only (no soap....soap can lead to overgrowth of harmful bacteria on the stallion's penis) !!
• Take cultures of the urethra before and after ejaculation, and the penile shaft. 
  • Culture the fossa, corona, etc. if there is a concern or a problem. 
  • You can expect no abundant pathogens, and there should be only garden types (mixed culture) seen on the cultures. 
  • You should not get a pure culture of anything.
• It is advisable to pull the shoes if at all possible to avoid injuring the mare or any of the collectors.

The semen collection

Click the movie icon to see a video of semen collection in the stallion.

• All the handlers should be on left side and everyone is advised to pull to the left if there is a problem.
• The entire collection has to be a choreographed effort by everyone involved in order to get a sample and keep everyone safe.
• Approach the mare at an angle and allow the stallion to mount the mare.
• Let the stallion thrust and guide or allow the stallion to insert his penis into the AV.
• Gently touch the ventral penis and feel the urethra for the ejaculatory pulses. Others can watch for the flagging of the tail that indicates ejaculation.
• After the sample is collected, keep open of the AV upright to avoid spilling the semen out.
• Immediately drain the water out to prevent the continued exposure of the sperm cells to hot water. The water jacket also forms a 'dam' that will prevent the semen from draining into the collection bottle.
• Protect the semen from light and cold shock and then begin to analyze it as soon as possible.

Semen analysis

• The sperm cells may make large circles due to normal abaxial midpiece, but this is normal for stallion semen.
• If an in-line filter was not used during collection, remove the gel with sticks or by pouring through a filter or some gauze.

• Use individual longevity tests for motility as well as looking at fresh samples.
• Motility - immediately examine raw and extended samples.
• Make sample consisting of a raw (neat) sample and samples extended 1:1 and 1:4.

• Keep the samples at room temp and prevent light or air contact.
• Estimate motility hourly until the motility is less than 10 %.
• The goal is to have at least 10% at 6 hours.
• If the motility is < 10 % after 3 hours, the stallion generally has poor fertility.

• Check the pH immediately with a pH meter.
• Normal pH is about 7.
• A higher indicates soap contamination, urine contamination, inflammation, or problems with the accessory sex gland fluid.

• A genital exam is best done after semen collection, because the stallion will be much calmer.
• Make sure the penis is free from:
• Summer Sores (Habronemiasis).
• Sarcoid.
• Melanoma.
• Coital Exanthema (Herpes).

• Check for edema of scrotum and adhesions between the testes and scrotum that will inhibit thermoregulation.
• Testes
• A stallion must have two!!
• Average size is 9x6x5 cm.
• The total scrotal width (TSW) should be > 80mm.
• Ultrasound exam is best
  • Measure height, width, length of testes to determine testicular volume
  • Examine testicular parenchyma, epididymides
  • Best way to examine scrotal contents (hydrocele, hematocele, hernia)

• The consistency should be the same throughout.

• The head is anterior, the body is dorsolateral and the tail is posterior.
• The orientation of the epididymis can help determine if a testicular torsion exists. Transient torsions are fairly common, and does not cause any clinical problems.

Rectal Palpation

• There are few problems diagnosed by rectal palpation and the risk of routine exams probably does not justify the rewards.
• You can check the accessory sex glands, which include the prostate, ampullae, and seminal vesicles.
• You can also palpate the inguinal rings for hernias.

• A second semen collection should be performed one hour after the first collection.
• The second collection, in relation the first, should have:
• Same Volume.
• 60 % as many cells
• Same motility
• A pH that rises slightly.
• If this relationship between the first and second ejaculates is not seen, then one of the ejaculates is not representative !!!

• The stallion must have normal libido, normal behavior and a normal gait.
• He should have 2 scrotal testes and there should be no scrotal or penile abnormalities.
• The testes should have at least 80 mm TSW.
• The cultures from the penis and urethra should have inconsistent bacterial types, and there should be fewer colonies after the first ejaculation. There should be no pathogens.

• The stallion should not have contagious equine metritis  (CEM) or EIA.
• If the stallion does not pass, recheck him again at a better time in the season or recheck him in 60 days.

Stallion management

• A stallion produces sperm cells seasonally, just as the mare cycles seasonally.
• Therefore, the season may affect the results of the BSE.
•  

• Lower fertility with some stallions
• Lower fertility with prolonged shipping times
• May be increased cost realized due to processing semen
• May be increased cost due to lower per cycle pregnancy
• Requires additional equipment and training for semen processing
• Requires better mare management
• Requires good stallion management
• Requires good communication between all parties
• Requires advance planning, semen may not be available every day

• Factors influencing success with transported semen
  • Pregnancy rates with transported, cooled semen are similar to those obtained after using fresh semen, provided mare management, semen quality and semen handling are good and shipping times are relatively short (< 24 hrs). Shipping times greater than 24 hrs are associated with some degree of reduction in pregnancy rates, possibly as much as 50%. For these reasons, proper techniques of semen evaluation, extending and packaging are essential.

• Semen quality / stallion fertility
• Motility, concentration, volume and morphology before extending
• Quality after storage / shipment
• Type of extender used
• Concentration, dilution ratio of extended semen
• Type of packaging system used
• Cooling rate
• Lowest temperature reached
• Ability to maintain stable temperature
• Duration of transport
• Number of normal, motile sperm inseminated
• Timing of insemination: ability to predict ovulation, use of hCG or GnRH analogues to induce ovulation and avoid need for repeat shipments
• Mare management / fertility

• USE OF SEMEN EXTENDERS AND SHIPMENT OF FRESH, COOLED SEMEN

   

  • The shipment and use of fresh cooled semen is experiencing increasing popularity. Restrictions enforced by the various breed associations are the main factor preventing its widespread use. Semen extenders are an essential component of fresh cooled semen.

• Semen Extenders

• Semen extenders are an important adjunct to an artificial breeding program. Use of semen extenders makes it possible to ship semen overnight while preserving fertility. Their beneficial effect is also used at times in natural breeding by infusing the extender into the uterus in conjunction with mating.
• When an extender is added to fresh semen, it is not unusual to see an initial increase in motility. As time passes, motility of the extended semen remains higher and is maintained longer, than the motility of the unextended, or raw, sample. In addition, semen extenders containing antibiotics help to reduce the contamination introduced into the mare's uterus at breeding. For these reasons, semen extenders may improve the fertility.

• Composition

• Semen extenders provide substances for the metabolic activity of the spermatozoa, buffer against changes in acidity and protect against cold shock. Glucose is the primary nutritive component of most extenders. Depending on the particular recipe, egg yolk or milk normally provides the protective effect against cold shock. If milk is used, either half & half, which is heated in a double boiler and the scum removed, or nonfat dry skim milk are usually used. Nonfat skim milk is very easy to use, however, only brands without added preservatives are suitable. Most commercially available extenders rely on non-fat dry skim milk as a base. Antibiotics are usually added to the extender to inhibit growth of bacteria in the semen during storage. Studies indicate that the antibiotic polymyxin B is not suitable for storage of semen, therefore it is not used in extenders for transported semen. Although other studies have indicated slight differences in motility after storage with various antibiotics, from a practical standpoint antibiotics such as ticarcillin, gentamicin or amikacin all give satisfactory results and are commonly used.
• Osmolarity and acidity are critical factors in the preparation of an extender. After preparing an extender, both pH and osmolarity should be checked before use. If instruments are not available to check pH and osmolarity, the extender should be tested to verify that sperm viability is maintained before the extender is used for shipping. Some antibiotics may significantly alter the pH of the extender and sodium bicarbonate must be added to restore it to a suitable range.
• Semen extender may be prepared in large quantities and then frozen and stored in smaller aliquots, such as 100 ml, for later use. Properly stored, the semen extender will be preserved for 3 to 6 months and reduce the need for frequent extender preparation. Commercially available extenders are very easy to use and are formulated to provide the correct pH and osmolarity. They usually consist of a packet of dry ingredients and a small vial of diluent which are mixed together at the time of use. Most are available in convenient 100 to 125 ml sizes so that extender is made as needed rather than pre-made and frozen.

• Dilution Ratio

• Sperm will quickly metabolize available substrates in seminal plasma so motility will decrease rapidly. Therefore, after the semen is collected, it should be mixed with extender as soon as possible, preferably within 10 or 15 min. Ideally, the ratio of semen to extender should be at least 1:2, and a ratio of 1:4 or 1:5 is preferable. Nevertheless, it is the final concentration of sperm in the extended sample that is the critical factor. Longevity of the sperm cells is maximized if extender is added to give a final concentration of sperm cells of 25 - 50 million/ml. With an adequate extender at a proper dilution, sperm will retain their fertilizing capability for up to 24 hours at room temperature (20^oC), depending on the individual stallion. Therefore a semen:extender dilution factor should be calculated based on the initial concentration of sperm to give a final concentration of 25 - 50 million sperm/ml before shipping.
• For example:

  • Concentration at collection = 267 million/ml

  • Desired concentration = 50 million/ml;

  • 267 / 50 = 5.3 total parts semen + extender

  • 5.3 total parts - 1 part semen = 4.3 parts extender needed

  • Therefore, use 5 parts extender : 1 part semen to give a final extended concentration of 45 million sperm / ml
• If a stallion provides an ejaculate with a low concentration, so that dilution at a 1:4 ratio, for example, would result in the final concentration being less than 25 million/ml, centrifugation is recommended. Centrifugation can be used to concentrate the semen so that dilution at a recommended ratio can be achieved while maintaining a concentration of 25 - 50 million/ml.
• Recommendations for centrifugation are to begin with a force of 500 X g for 10 minutes. Less time or fewer g’s will result in less damage to the sperm from the force of centrifugation but a softer pellet and more viable motile sperm in the supernatant that will be discarded. Centrifugation for a longer time or at higher g’s will achieve a better recovery and minimize sperm losses in the supernatant but result in more damage to the sperm cells. Centrifugation technique can be altered within a range of g’s and times to achieve good recovery while minimizing cellular damage.

• After centrifugation, the supernatant is removed and discarded. Studies have shown that a small portion (a minimum of 5%) of the seminal plasma must be left with the sperm to preserve viability. The pellet is then resuspended using sufficient extender to achieve a final concentration of 25 - 50 million/ml.
• Preservation of semen quality depends to a large extent on the initial quality of the semen, and varies from stallion to stallion.