Semen Evaluation



 

• Gross motility is examined first. 
  • Mix the semen sample with a wooden stick, as motile sperm cells will try to swim upward and dead cells will settle to the bottom. 
  • For gross motility use 2 wooden sticks to place a drop of semen on a warm slide. 
  • Do not use a cover slip and examine the cells under a 10X objective. 
  • The motility is judged by the swirling motion of the sample. 
  • The swirling pattern will definitely indicate that there are cells alive. 
  • The swirling looks like currents and eddies (like a fast motion weather map). 
  • Assessment of individual cells cannot be made as live cells will carry the dead cells. 
  • The gross motility will tell you though that the cells are alive, and if the cells are dead when you examine them for individual motility there has been a handling problem.  
  • Gross - swirl pattern in bull, ram and buck.

  

  Gross Motility

 

• Individual motility is examined next. 
  • Individual motility checks for the progressive movement of the sperm cells. 
  • Make the sample by placing a drop of diluent (saline or Na citrate) on a warm slide. 
  • Place a small amount of semen into the saline. 
  • You want about 10 cells/high power field in order to accurately estimate the number of cells that are progressively moving across the field. 
  • Then place a warm cover slip on the drop. 
  • Examine the sample under high dry (40X) power. 
  • You must examine the sample quickly as the motility changes very rapidly with heat, light, and cold.

• The motility is a very subjective measurement and is affected by many things, such as diluent (it may be old and hypertonic etc.), cold, the glassware, urine, soap, prostatic fluid, seminal pH, and ion composition.
• Computerized sperm motility computers (CASA) are available for $30-50000
  • Objective, but they probably do not tell us that much more than by eye.

    

    Individual Motility
    
    

    Sperm morphology
• Morphology is usually examined with an eosin-nigrosin (Society for Theriogenology) stain (background stain) to highlight the cells. 
  • The slide is made by painting a drop or line of stain on a warm slide, then using a wooden stick to place a small amount of semen into the stain; the semen and stain are mixed using another slide, then slowly push the second slide through the stain and across the first slide while pressing firmly down. You should be making a 'bad' blood smear. 
  • The goal is to get a dark background, as the stain is a background stain and is not intended to stain the cells. 
  • In fact, some cells will stain red, but this makes no difference in our evaluation. 
  • You want the cells to be close, but not overlapping. 
  The final slide should have dark and light areas that allow you to view different colored backgrounds as needed when examining the slide.

• Examine the cells under 1000X (oil) to fully assess the morphology.

• Count 100 cells and, in a practice situation, you can just differentiate normal from abnormal cells. 
  • Using eosin-nigrosin all the cells will appear flat, as if looking at your hand. In order to do a full spermiogram, differentiate the abnormalities by type. 
  • A phase contrast microscope can be used, but is rare in a practice situation. 
    • A phase contrast mount is made by placing a small amount of semen in formal-buffered saline to kill and preserve the cells. 
    • Then place a drop of the formal-buffered saline sample onto a slide and place a cover slip over the drop. 
    • You need a phase contrast microscope to examine the specimen. In using the phase-contrast microscope, match the phase ring with the power that you are using. 
    • The phase contrast acts like a 'stain', however the cells will float by instead of lying flat. 
    • The cells now look like your hand from the top and side views.

Abnormalities  

• Abnormalities are classified as primary and secondary. 

  • Primary abnormalities are thought to arise in the testes, whereas secondary abnormalities arise in the epididymis or ejaculate. 

  • Secondary abnormalities may be just as serious primary. 

  • Primaries abnormalities decrease through transit and secondary abnormalities increase through transit.  

• Major and minor may be a better classification. 

  • Major problems cause Early Embryonic Death or prevent fertilization. 

  • For example, an acrosome problem prevents zona entry, and a nuclear problem leads to nonfertilization or EED.  

  • Minor problems such as tail abnormalities etc. stop sperm movement, so the sperm cell cannot get to egg. 

• Some abnormalities are compensable and some are non-compensable. 

  • A non-compensable abnormality gives the animal a poor prognosis for any recovery. 

• Problems with sperm cell examination. 

  • The light microscope cannot always detect abnormalities.  

  • Abnormal sperm cells indicate cellular damage, but the relationship to fertility is only circumstantial.
    
    
    
    Normal Sperm Cell above

            Chart of Sperm Cell Abnormalities below
  

                                       Click to enlarge
  
  Abnormalities of the sperm cells include:
  

• Decapitated sperm (not pictured)- the basal plate is defective; tails move (wrap around drops); 100% cells involved; occurs in epididymis (secondary)  

• Loose heads (not pictured)- these may be increased on the first ejaculate (rusty load) because the tail attachment is frail. 

• Knobbed or flat acrosome (not pictured)- the acrosome folds over itself of the apex of the acrosome is knobbed or flattened. When 20% of the cells have acrosome problems the result may be infertility in the bull. This condition may be hereditary in Charolais, Hereford, and Holsteins.
   

• Wrinkled acrosome - this may reflect a nuclear problem which prevents zona attachment by the sperm cell. It is a rare condition.  

• Pyriform and tapered heads - the nuclear material is poorly distributed. The defect may be subtle.

• Giant or small heads - This a nuclear problem. If the head is twice normal size the cell is a giant cell.

• Nuclear vacuoles - These distort the shape of the head.

• Diadem defect - With this you see invaginations in the nucleus, mostly by the post nuclear cap. The pit lacks DNA. The condition may be associated with stress in bulls and may come and go as stress changes.

• Dense proximal droplets - This arises in the epididymis and indicates maturation problem.
• Stump tail defect (not pictured) - this is an axonemal problem. It looks like a cytoplasmic drop and has a poor prognosis. The incidence increases with age. Midpiece defects - You see lumps on the midpiece that can be confused with proximal drops. Coiled mainpiece - The mainpiece is coiled within the plasma membrane.

• Dag defect - This is a sterilizing defect that occurs in the epididymis so is it is actually a secondary abnormality, but it is a major defect. The condition is inherited and the axoneme is disrupted (fibrils and helix). You see split, shattered, or fractured midpiece. The tail may coil and the motility is low.
• Coiled midpiece (not pictured). This is an epididymal defect, but is a major defect. The midpiece problems (not Dag).
   
• Abaxial midpiece - the implantation fossa is defective. You may see abaxial or double midbpieces; There is generally a low incidence and fertility is not affected; (normal in stallion).

• Coiled mainpiece - The mainpiece is coiled within the plasma membrane.

• Teratospermia - the entire cell is degenerative.
   

• Bent tails - the bend in the tail may include a droplet which may be in the membrane.

• Physiologic (distal )droplet - some consider this a minor defect, but in fact it may be a major defect. These cells do not freeze well because the water in droplet crystalizes and ruptures the cell membrane.

Temporal progression of sperm abnormalities after 4 days of scrotal insulation.

Days after scrotal insulation	Sperm abnormalities in the ejaculate	Days return to normal
7-11	Distal midpiece reflex, Proximal Droplets	40
11-15	Mitochondrial Sheaths, Detached heads	35
18	Knobbed acrosomes	25
20	Nuclear Vacuoles	22
22	Pyriform heads	20
23	coiled principal piece	19
42	normal	0

• In bulls and rams, the sperm count is estimated by measuring the scrotal circumference. The scrotal circumference measurement is used because electroejaculation does not give a physiologic sample that can be reliably counted. 
• Hemacytometer method
  • To count the concentration of semen sample a hemacytometer can be used. 
  • The hemacytometer is loaded with a 1:100 dilution of semen (see below how to make a 1:100 dilution, 
  • a hemacytometer coverslip is placed over the chambers
  • the chambers are filled  
  • the sample allowed to settle, 
  • all the sperm heads in the middle big square (the square with 25 smaller squares (....and another 16 squares within each of these 25) within the triple lines) are counted. 
  • The number of sperm heads counted in a single chamber is multiplied by 10⁶ to give the concentration of cells/cc. 
  • Both chambers of the hemacytometer should be counted and the numbers should not differ by more that 10%. 
  • The coverslips are expensive so do not break them or dispose of them!
  • Sound confusing...look at the diagram.
    

• The hemacytometer grid has 9 large squares. The central square has triple lines around it. Inside the triple lines are 25 smaller squares (also bounded by triple lines). Within each of the 25 squares are 16 squares (only one of the 25 cells is illustrated having the 16 smaller squares. This is illustrated below, however the triple lines are only shown on the outside edges of the middle big square. 

Here is an actual picture of the grid of the 25 squares in the middle big square. They are all bounded by triple lines. Count every sperm head bounded by triple lines.

Here is a picture of one of the 25 squares with the 16 squares inside.

For example, if each of the dots represented a sperm head, the count on the grid below would be 13 x 10⁶ cells/ml.

• The 1:100 dilution can be made using a Unopette diluter or a hand dilution. 
• The Unopette is easier, but sometimes the cells will clump, so an accurate count cannot be made.
  •  If using the Unopette, first puncture the bottle with the sharp point, then draw up the semen via capillary action into the capillary tube. 
  • The bottle is then squeezed and the capillary tube inserted into the bottle and the semen sucked into the bottle. 
  • You can turn the capillary tube over and use it as an applicator to load the hemacytometer chambers.
  • We no longer use the Unopette at LSU

• A hand dilution is made by:
  •  diluting 1 part semen with 9 parts formal-buffered saline to make a 1:10 dilution, 
  • then taking 1 part of the 1:10 dilution and adding 9 parts of formal-buffered saline to make a 1:100 dilution. 
  • This is a little more time consuming, but the cells tend not to clump, so you get a more accurate sperm count.
      

  Click on the above diagram to see a slide show demonstration.

• A spectrophotometer can be used to count stallion semen (and possibly dog semen if the correct software is used). 
  • Basically, the machine measures the amount of light that passed through a sample and calculates the concentration of cells that the density reflects. 
  • Using our machine:
    • A blank tube is loaded with 3.42 ml of formal-buffered saline 
    • The blank is inserted into the machine with the clear sides on the left and right. 
    • The machine passes a beam of light through the clear sample and determines that 100% of the light is passing through. 
    • Then, 180 µl of semen is added to the tube, the tube mixed and reinserted into the machine. 
    • The sperm concentration is then calculated based on the percent of light that is transmitted through the sample. 

    • Any dirt, blood or other contamination it the sample may adversely affect the amount of light transmitted and result in an erroneous concentration reading.

   

• SpermAcue
  • Spectrophotometric method
  • Canine and equine

Total sperm numbers.  

• NucleoCounter
  • Cell membrane disrupted
  • Dye stains DNA
  • Digital image created
  • Cells counted
  • Can also be used to count intact membranes by not disrupting the cells first

  

  

• Multiply the concentration X the volume to give the total number of cells in the ejaculate.
• Take that number and multiply by the percent progressively motile cells to get the total number of progressively motile cells. 
• Take that number and multiply by the percent normal cells to get the total number of normal, motile cells.
•  For example: 100 cc X 50million cells/cc = 5 billion cells 5 billion cells X 50% motility = 2.5 billion motile cells 2.5 billion motile cells X 50% normal = 1.25 billion normal, motile cells.
• Total sperm numbers are used in stallions and dogs, but not in bulls, rams or bucks.

• 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.

• Determination of insemination dose

• Prior to shipment of semen from a stallion (and periodically during the breeding season) it is advised to do a trial run with the chosen shipping system and determine the motility after 24 and 48 hours of storage. This allows you to determine the "recovery rate" and make adjustments in the number of sperm shipped so that an adequate insemination dose will be provided when the semen reaches its destination.
• In addition, it is advisable to maintain a record of the collection data. Such information may help in determining the collection or shipping frequency, shipping dose and number of doses to expect from an average collection.
• The recommended insemination dose is usually 500 million progressively motile (normal) sperm, although acceptable pregnancy rates can be achieved with as few as 250 million normal, progressively motile sperm. The volume of the inseminate is not critical. Although pregnancy can be achieved with very small volumes, the recommended minimum volume used for insemination is usually 10 ml. Usual insemination doses with fresh cooled semen range from 20 to 120 ml. Although some veterinarians are reluctant to inseminate volumes greater than 60 ml, studies have shown no decrease in fertility when larger volumes were inseminated, provided the inseminate was not real dilute.
• To determine the volume of semen to package for shipment, divide the desired number of progressively motile sperm (usually 500 million) in an insemination dose by the product of the concentration times percent motility at the end of the storage period. For example, if we have extended our semen to a concentration of 50 million/ml and our motility after transport is 40%, we should package at least :
• 500 / (50 X .40) = 500 / 20 = 25 ml. Unless a stallion is in very high demand it is best to package more than the minimum amount needed. For example, with the above stallion, packaging 50 ml would insure that more than adequate numbers of sperm were available for fertilization of the oocyte. Furthermore, a more conservative method is to include percent normal morphology into the equation. For the stallion above, if normal morphology is 70%, the equation becomes:
• 500 / (50 X .40 X .70) = 500 / 14 = 35 ml

• Semen Packaging
  • The pre-warmed extender should be added slowly to the semen. The extended semen should be placed in a container such as a Whirl-Pak bag, from which air can be excluded, and sealed. This container should then be placed in a second container from which air is again excluded and the second container also sealed.
  • It has become commonplace for some people to place two insemination doses in the shipping container, intending one to be used upon arrival and the other to be used the following day. In fact, there is no physiological basis for such practices. The ideal place to store spermatozoa is in the mare’s oviducts. Man-made semen transport devices are a means to transport semen from the stallion to the mare without transporting horses. They are not meant to take the place of the mare as a site of sperm storage until fertilization. All semen received in a transport device should be inseminated into the mare at the time of arrival. This should be kept in mind when preparing the semen for shipment.
  • Ideally, the semen should be extended and placed in the cooling device within 10 minutes of collection. The rate at which extended semen is cooled is critical. If the cooling rate is too fast or too slow, sperm viability is decreased. A cooling rate of -0.05 to -0.1 C/min is desired between 20 and 5 C. Ideal storage temperature is 4-6 C.
  • Numerous types of containers for fresh, cooled semen are commercially available. Reusable containers such as the Equitainer as well as inexpensive disposable systems are in use worldwide. The systems vary in their cooling rates, minimum temperature reached, time required to reach the minimum temperature, length of time they are able to maintain the minimum temperature, etc. Performance of the different systems also varies depending on environmental conditions. Some of the disposable systems such as the Equine Express and BioFlite compare very favorably with the Equitainer system, while others such as the ExpectaFoal compare less favorably, reaching temperatures below 0 C.
  • Some reports indicate sperm viability is decreased due to contact with the rubber plunger on syringes. Syringes constructed of all plastic are therefore recommended by some researchers. In actual practice, however, the extended semen is not in contact with the plunger very long and the numbers of sperm cells being inseminated are great enough that toxicity from the syringe is probably of little clinical significance. If semen is held in syringes for any length of time, however, as in some types of shipping devices, syringes of all plastic should be used.
  • An information form should be included with each shipment. Minimum information required on the form includes stallion identification, date of collection, concentration and volume of inseminate shipped (i.e. numbers of sperm), initial motility, type of extender used, numbers of doses shipped and any special instructions.
  • Prior to shipment of semen from a stallion (and periodically during the breeding season) it is advised to do a trial run with either shipping system and determine the motility after 24 and 48 hours of storage. This allows you to make adjustments in the number of sperm shipped so that 500 million motile sperm will be provided when the semen reaches its destination.
  • Use of some of the more common packaging systems are described in more detail:
    • EQUITAINER

      

      • The Equitainer system is a very durable, hard plastic insulated container. In the center of the Equitainer is a well. The system is designed to cool a volume of 120 to 170 ml of fluid at the optimum rate. The extended semen sample is nestled in ballast bags in a plastic cup so that the total volume of the semen plus the ballast bags is between 120 to 170 ml. Each ballast bag holds 60 ml of a colored fluid. Ballast bags should be warmed in an incubator (37^oC) for 4 hours before use. The plastic cup containing the semen is placed into the "isothermalizer". One or two (depending on the model of Equitainer) specially designed freezer packs are placed in a plastic bag and loaded into the well of the Equitainer. The isothermalizer is then loaded into the well on top of the freezer packs. Records should be enclosed that identify the source of the semen and for the person breeding the mare to fill out. The particular forms may differ depending on the breed involved. The container is then closed and latched. It is a good idea to seal the container so that it will be evident if any tampering occurs.
      • A sample of the extended semen that was packaged in the Equitainer should be kept in a similar manner for evaluation 24 hours later, as a quality control. If a second Equitainer is not available, the cooling and storage procedure can be simulated by placing a sample in a Whirl- Pak bag which is then placed in a polypropylene cup. The cup is then placed in a water bath of approximately a pint of water in a container about 6 inches in diameter, which has been prewarmed to 37^oC. Place the whole assembly into a refrigerator set at 5^oC. After a 24 hour interval, warm the semen to 37^oC and assess motility.
      • Various models of the Equitainer system are available. Models differ somewhat in the length of time they will maintain the semen chilled and whether a lead shield is present. It should be remembered, though, that shorter storage times result in improved fertility.
    • EQUINE EXPRESS

    

• Shipping
  • The use of fresh, cooled semen provides a number of advantages. It is much easier to ship a container of semen to a mare than to ship a mare and foal to a stallion. Not only is cost decreased by shipping semen rather than horses, but stress on the horses is greatly reduced also. Fresh, cooled semen allows more efficient use of a stallion, not only for shipping, but for temporary storage on the farm to reduce the frequency of collection. For example, a stallion can be collected, the ejaculate extended and a portion used to inseminate mares that day. The remainder can be cooled and used to inseminate mares 24 to 48 hours later. Shipment of semen increases the availability of superior stallions or stallions of uncommon breeds and allows a stallion to breed a greater number of mares in a season.
  • Some slight disadvantages are inherent in the use of shipped, fresh cooled semen. For unknown reasons, considerable variation exists between stallions in the ability of their sperm to remain viable during the cooling and storage process. For all stallions, however, fertility is generally higher if the storage period is shorter. If care is taken in the preparation of fresh, cooled semen and mares are managed well, pregnancy rates using fresh, cooled semen can be as high as those with a natural breeding program. An important consideration if semen is being shipped in to breed a mare is the advanced planning required. This may be complicated by the fact that semen may not be available every day of the week due to the work schedule of the shipping company. In addition, use of shipped semen requires good mare management. Good record keeping and a good teasing program are integral components of a successful breeding program. Ability to predict ovulation is essential in order that semen arrive before the mare ovulates, and to avoid repeated shipments of semen during a single estrus. Research has shown that breeding too long after ovulation results in decreased pregnancy rates and increased early embryonic death.

PREPARATION AND SHIPMENT OF FRESH, COOLED, CANINE SEMEN

• Semen is analyzed 

• Extension 

  • Appropriate semen extender is added to the ejaculate, usually 1:1. 

  • Semen extenders provide an energy source and buffers that enhance the survival of chilled sperm cells. 

  • Extenders can be obtained from commercial sources that are manufactured exclusively for extending canine semen (Synbiotics, San Diego, CA 92127; CLONE, Chester Springs, PA 19425; Camelot Farms, College Station, TX 77842; International Canine Semen Bank, P.O. Box 651,Sandy, OR  97055), 

  • Equine semen (Lane Mfg., Denver, CO 80231; IMV Intl, Minneapolis, MN 55430) work well and are much cheaper.

  • Homemade semen extenders can also be prepared, however proper laboratory techniques

  • The extender must be pre-warmed before adding it to the semen or the spermatozoa will suffer cold-shock. 

  • The prostatic portion of the ejaculate may have detrimental effects on the storage of canine sperm cells,

  • We found no detrimental effects on pregnancy rate or fecundity when whole ejaculates were extended 1:1 and inseminated after 24 or 48 hours of storage.

  • If the entire prostatic portion of the ejaculate is collected and the semen is extended, the volume of the resulting extended semen may be such that it cannot be completely inseminated without some vaginal reflux.

  • Our research at LSU has shown (EVSSAR Estoril 2007):

    •  Prostatic fluid and centrifugation have no effect on the membrane integrity of the cells when semen is extended in a commercial equine extender. 

    • Semen extended in a commercial equine extender and chilled should have the prostatic fluid removed before storage and it should be stored at a relatively high concentration for maximum motility.

    • If a sample does  arrive that has a large volume because the prostatic portion was included in the extended semen, centrifugation to remove the prostatic fluid yields progressive motility that is not different from removing the prostatic fluid before extension.

• Containers

  • Containers designed to ship semen can be obtained from many of the same sources that provide canine semen extenders. (Synbiotics, San Diego, CA 92127; CLONE, Chester Springs, PA 19425; Camelot Farms, College Station, TX 77842; Bio-Flite, Anaheim Hills, CA 92807; International Canine Semen Bank, P.O. Box 651, Sandy, OR  97055). 

  • The containers usually consist of a Styrofoam box, an ice pack, and container for the semen.

  • These commercially available semen containers maintain semen well enough that acceptable pregnancy rates result. 

  • Commercially available shipping containers offer a predictable, attractive and easy way to ship semen. 

  • Many of these containers are relatively expensive when compared to the disposable equine shipping containers. 

    • A commercially available (although no longer marketed) disposable equine shipping container allowed storage of extended canine semen for up to 48 hours and the resulting pregnancy rates were equivalent to AI with fresh semen.

    • We have used other brands of  disposable equine semen  shippers and have had good success in maintaining semen viability. 

    • The advantage of the equine shippers is their lower cost than the canine semen shippers