Semen Analysis and Cryogenics

Credits: Generously provided by Clover Williams and Katelyn Barber, with editing  by Louise Schofield, Guiding Eyes for the Blind, Yorktown Heights, New York, USA

We all have heard of breeders who believe they have a wonderful stud dog, only to find that he is not able to produce a litter. Or, that a wonderful dog has produced just one excellent litter, and has then died in an accident or from old age.  To maximize breeding success, proper handling of the stud dog and analysis of his semen is critical, and to preserve the excellent stud dog, cryogenics offers a solution.


5.5.1 Collecting and Handling Semen


It is essential that every time a stud is collected, whether it is for a routine evaluation, breeding or freezing, that it is a positive experience for the dog. Negative experiences can cause anxiety in the stud, and there are some cases in which studs can “shut down” and not ejaculate properly.


Semen collection will require two people, and the use of a teaser bitch, which should be in prime standing estrus. One person, the collector, should ready a collection funnel or artificial vagina, used to collect the ejaculate from the stud.  The bitch should be closely secured to a wall or post, and held still by a helper.  The stud should then be brought into the room and allowed to investigate the teaser bitch.  Eventually, the stud will mount the brood, and begin to thrust.  At this point, the assistant holding the brood must cover the brood’s vulva with her hand, to ensure penetration does not occur.  The collector can then encircle the penis with a lubricated, gloved hand, which will cause the stud to thrust harder and begin to ejaculate.  The first portion of the ejaculate, which is clear, should not be collected. Once thrusting slows, a milky white section, called the sperm-rich fraction, will be ejaculated.  This is the important part to collect, and will typically be from 0.5 – 2.0 ml.  The third fraction, which contains prostatic fluid, should only be collected if the semen is to be used for a vaginal artificial insemination (AI), where the extra volume is needed.


Extreme care should be taken when handling semen.  Cold and heat shock can occur within sperm, causing morphological defects.  Care should be taken to ensure that the temperature of the semen during the collection and processing phase remains as close to 33 degrees Centigrade as possible.  Try to process the semen as quickly as possible to prevent degradation to the quality. Chilled semen and frozen semen require specialized handling procedures.



5.5.2 Semen Analysis


Routine semen analyses and palpation of the testes and epididymi should be performed on all breeding males every three months at minimum. Ideally, a stud dog’s semen should be collected a few days prior to the peak breeding days to ensure that aged sperm are voided prior to actual breeding, and that semen quality remains high.  A routine semen analysis is best performed using a phase microscope, and should include:


  • Motility (movement) percentage


  • Speed rating


  • Consistency and clarity measure


  • Count based upon volume


  • Concentration of sperm in the ejaculate


  • Morphology (abnormal sperm) examination


  • Check for the presence of other cells (white blood cells, red blood cells, epithelial cells, etc)


A sperm concentration as low as 100 million sperm/ml can achieve good pregnancy rates, provided the sperm speed, motility and morphology are good. The normal sperm count for the large-breed dogs used at guide dog schools is typically 500-1200 million sperm per ejaculate, which equates to a concentration of 200,-,500 million/ml. Some colonies, however, record normal sperm counts as low as 300 – 400 million sperm/ejaculate.  Usable sperm should have a motility of greater than 70%, and possess the ability to have good forward movement.


Abnormalities can be an indicator of testicular degeneration or inflammatory problems in the prostate, epididymides or testes. Blood in the semen due to a ruptured blood vessel will normally not affect sperm function and fertility.  Abnormalities could also be caused by poor semen handling.



5.5.3 Ways to Share Your Stud with Others


Sharing semen among schools is an effective way to improve the quality of one’s colony, and can sometimes increase the rate of genetic improvement faster than using only your own studs. To learn more about genetic improvement through sharing studs, see Section 5.6.3, Global Solutions for Selective Breeding.   Schools that practice genetic selection may possess a stud that would be a great asset to another school’s breeding program.  In this situation, it would be beneficial to both schools to share that stud’s genetic material.  There are several ways to accomplish this:


  • Chilled semen


  • Stud loans


  • Frozen Semen


Chilled semen is an ideal way of using studs that live beyond driving distance from the bitch. The sperm-rich fraction only is collected into an extender such as Fresh Express™, which is available from vendors such as Synbiotics. After processing by slow cooling, it is shipped on ice to the bitch. Insemination can be done vaginally or transcervically. Volumes of 5 – 12ml can be used for vaginal AI, while transcervical or surgical AI requires that the total sperm dose is contained in only 1 to maximally 3 ml total volume.


Stud loans.  Small or developing schools may not possess the technology necessary to perform artificial inseminations.  In this case, the most feasible way to share genetics would be through a stud loan.  Stud loans involve shipping a stud to a different school for a specified period of time.  The receiving school can then perform natural matings with a genetically superior stud.  Shipping dogs, especially to other countries, requires a great amount of planning.  Customs paperwork, health certificates, and shipping arrangements must be made.  You must check with the receiving country to determine what quarantine periods or paperwork must be completed before the dog will be granted entry.


Frozen semen.  Frozen semen is the preferred method of utilizing semen from dogs from other countries or at later time periods when the stud dog is no longer available. Insemination can be done most successfully with transcervical or surgical procedures.  Due to the highly compromised state of frozen semen, litter size and conception rate is less than can be achieved with fresh semen matings.  Conception rates of approximately 75% and average litter sizes of five puppies can be expected when using frozen semen.



5.5.4 Cryogenics – Preserving Semen through Freezing


Cryogenics is the science of the effects of extremely low temperatures. Cryogenic storage at very cold temperatures in tanks of liquid nitrogen (-196 Celsius) can preserve indefinitely a range of biological materials, from tissue and blood to embryos and semen.


A few of the larger U.S. guide dog schools maintain their own cryogenics facilities to preserve and store guide dog semen. Commercial organizations provide cryogenics services around the world, and there are veterinarians specializing in reproduction who maintain cryogenics facilities for collection and storage.


Why Use Frozen Semen?


Frozen semen one of the most valuable tools available to create genetic improvement in small or developing schools. However, it does require significant planning and effort to obtain frozen semen, and should be considered long before the brood comes into heat.


There are several reasons to incorporate frozen semen into a breeding program, including:


  • The ability to breed broods with a genetically superior stud located on the other side of the world


  • Reduce inbreeding coefficients


  • Frozen semen can be shipped worldwide, removing distance as a barrier in the search for a superior stud


  • Once a school is equipped to handle frozen semen, it requires less expense and maintenance than housing a visiting stud



Frozen semen allows a stud’s genetic material to be preserved and easily shipped, making it easy for schools to share their best studs with others.  See Section 5.6.3, Global Solutions for Selective Breeding, for more information on how importing semen from other schools that practice genetic selection can improve your colony.



Storing Frozen Semen  


Once the semen to be frozen is collected from a dog, it is added to an extender and quickly brought down to a low temperature, and is then stored permanently at extremely low temperatures in a special tank that is filled with liquid nitrogen (LN2). The semen is contained in thin “straws” that are put in separate canisters, which then go into the tank.


Maintenance of the semen storage tank is vital. As liquid nitrogen evaporates over time, it is necessary to replenish the tanks with small amounts of LN2 each week. Normally, companies selling various gases to hospitals or welding companies will deliver liquid nitrogen.


It is imperative never to let the storage tank run low or out of liquid nitrogen or else the semen will be ruined. On a weekly basis, measure the LN2 level in the tank with a yard/meter stick. Be alert to a significant change in the amount of liquid nitrogen. If it drops dramatically, this is an indicator that the tank may be failing. Tanks need to be replaced about every fifteen years, although tank failures due to cracks from defects or mishandling can shorten the tank life. In addition to measuring weekly, almost daily checks should be performed by observing the outer tank for evidence of frost. If a tank is operating correctly, the exterior should be at ambient temperature (NOT cold), with no frost present. Some tanks can be equipped with temperature sensors at an additional cost.



5.5.5 Insemination of Frozen Semen


See Section 5.4.1, Insemination Techniques, to learn more about inseminating a brood with frozen semen.  Transcervical insemination or surgical insemination are the recommended procedures to use.  For schools that have not previously used frozen semen, it may be very helpful to enlist the help of a veterinarian who specializes in reproduction, or an experienced breeder from another school, in order to provide training and insight on the insemination process with frozen semen.  Careful semen handling should be of utmost concern, and semen should always be evaluated under a microscope before insemination occurs.


When inseminating with frozen semen, timing of the AI is crucial, as the thawed sperm do not live as long as fresh sperm. Generally, two inseminations are done by transcervical insemination at LH days 4 and 6, or days 5 and 7. If the semen quality is poor based on the post-thaw quality, inseminations would include more straws or matings on LH 4, 5 and 6.


Note:  The facility that froze the semen will provide information on sperm dose (number of straws per AI) and the technique for thawing the semen. Freezing facilities use different freezing systems. For optimum results, it is important to follow the recommendations specific to that system.



5.5.6 Shipping


There are no hard and fast rules on canine semen shipping, as conditions vary so markedly from country to country. Some countries require numerous blood tests and veterinary exams at specific time periods before or after semen collection. This requires planning and some additional costs for the school doing the freezing.


The U.S. Department of Agriculture runs an excellent website for identifying the current regulations for shipping canine semen internationally: 


Some countries, however, do not have regulations in place for the importation of canine semen. Note that canine semen is treated as “Animal Export” rather than “Animal Product” which deals with meat, hides, etc.


When planning to export semen to another country, it is imperative that the import regulations are complied with completely. The recipient of the semen should be responsible for providing the shipper with all current importation regulations.


If at all possible, use “vapor/dry” shipping containers rather than “wet” ones. Vapor/dry shippers wick the liquid nitrogen into an absorbent liner, which stores it as vapor rather than liquid. Therefore, there is no LN2 to spill, and the container is classified as a “non-dangerous good”.  Using a commercial shipping company can also be a large help during the shipping process to assure that all paperwork and requirements are completed and met, and the package arrives in a timely manner.