Semen Collection and Tips for Successful Breedings
The following interview was originally released as a podcast on February 27, 2014. This is an abbreviated transcript. Dr. Schultz elaborates on his answers in the recorded podcast, so please listen to learn even more.
Dr. William Schultz received his DVM from Michigan State University in 1973, went into private practice and opened his companion animal practice in the fall of 1974. Dr. Schultz is a well-recognized expert in canine reproduction, a member of The Society for Theriogenology and The Theriogenology Foundation, and is a frequent speaker at veterinary conferences, veterinary associations and national specialties. Dr. Schultz has lectured and published articles on transcervical and surgical inseminations using fresh, chilled and frozen semen.
This podcast is part of a special series of podcasts on canine reproduction in partnership with our corporate alliance, Zoetis.
AKC CANINE HEALTH FOUNDATION (CHF): Let’s talk in general terms about canine artificial insemination and ovulation timing. In your opinion, when are they of greatest benefit to breeders?
DR. William Schultz: Artificial insemination has been done for many years, but the ability to freeze semen and chill and ship semen has dramatically changed the ability to do planned breedings with incredible success. Vaginal, transcervical and surgical inseminations are now the norm for many kennels. Ovulation timing has changed from a semi quantitative vaginal cytology to progesterone testing and the gold standard for timing – serum LH – leuteotrophic hormone. We will discuss testing and breeding methods later in the podcast. The benefits to breeders are nothing short of incredible. It is now possible to successfully breed with semen from almost anywhere in the world. The bitch does not need to leave home to be bred decreasing the stress related to travel and spending time in a strange environment waiting for her to stand and breed. Travel can decrease conception due to stress and with the cost of travel today staying home has financial benefits to the breeder as well.
CHF: We can start this conversation with the collection of semen used in artificial insemination. What should the environment be like for semen collection from a stud dog and what should you have on hand?
Schultz: In our clinic we collect in the same exam room so that the stud will become familiar with this room. We do not vaccinate or do other procedures with studs in this room. We also have a breeding rug. This rug is rubber backed to allow for non-slip footing and is only washed as needed. The stud is brought in the room and allowed time to acclimate and sniff around. Large breeds are collected on the floor and smaller breeds are collected on the examination table. The rug is moved to the table to assure non-slip footing. The room is not centrally located which allows for a quieter atmosphere. In a kennel the collection area should be isolated as much as possible - sometimes a quiet store room area can be used. Keeping the outside distractions to a minimum will always be helpful. We use a disposable artificial vagina for the collection. These are cone shaped with the end cut off to allow the placement of a 15ml centrifuge tube. The tubes may be changed during the collection to separate the semen fractions. The wide end of the cone may be folded to the outside to adjust the length to accommodate different size stud dogs. Lubricants are not needed. This is a much better method than collecting into a baggie. The cone will fit firmly stimulating a tie and the studs are much more receptive to a complete collection. A teaser bitch is extremely important and in some males she may need to be in full standing heat. We have had inexperienced males not release sperm rich fraction without a standing teaser. This may lead to an incorrect diagnosis of infertility. Avoid wearing white coats, act relaxed, friendly, and non-threatening. The collection may be performed on the floor or on a table, depending on what the stud is used to. If there is a particular item that the stud associates with breeding, such as a rug or breeding rack, have the owner bring it along. All equipment must be at room temperature.
CHF: Why do you need to have a teaser bitch in the room and if you don’t have access to a female in heat is there anything else you can do?
Shultz: In almost all cases the teaser will significantly increase the ease of collection and the volume of the collection. It is the single sperm that completes the insemination but it takes millions to billions of sperm to get to that point. If no teaser is available we use swabs that were used for vaginal cytology in previous bitches. We keep the swabs in a baggie in the refrigerator and allow the male to sniff the swabs. These swabs may be frozen but we find that the refrigerator works well and we replace swabs monthly. If a bitch is used that is not in heat the swabs may be rubbed near her anal and vulvar area simulating a bitch in heat. It is important to closely control the non-cycling bitch because she may become very aggressive if male tries to mounts her. Zoetis makes an artificial pheromone called Eau d’Estrus that may be helpful if swabs and a teaser are not available. If at all possible, have an estrus teaser bitch available that is approximately the same size as the stud dog. Bring the bitch into the room. A handler or technician should be available to support the bitch in a standing position and restrain her if necessary. If that is not possible, use an anestrous bitch, restrained in standing position. It may be useful to keep some swabs of estrus vaginal secretions frozen. Thaw these by dipping them in warm water, and then wipe them on the vulva of a teaser not in season. Often 30-50% more sperm cells will be released when a suitable teaser is used. Many dogs may have semen collected without a teaser bitch, but semen quality is generally better when an estrus teaser bitch is present and the stud's libido is highest. An alternate training tool is a pheromone such as Eau d’ Estrus.
CHF: Is there anything that should be done before a dog is brought into the room for semen collection?
Shultz: Taking the male for a walk and allowing him to urinate before the collection is helpful. This should be done completely away from the teaser if possible.
CHF: Once the dog is brought into the room how should he be handled?
Shultz: Again, the male is allowed to acclimate and to interact with the teaser before any attempt is made at collecting. The least number of owners and assistants in the room is also necessary to decrease the level of distraction. However, a good stud dog will not be bothered as long as a teaser is present.
When performing semen collections, take care to make the stud dog feel as comfortable as possible.
CHF: Can you tell us how semen should be properly collected?
Shultz: With the bitch in position, bring the stud into the room. Allow him to familiarize himself with his surroundings, with the bitch, and with you.
If the teaser is not to be bred, be careful at this time that he does not rapidly mount and tie the bitch.
I sit in a low chair to allow rapid movement while the stud is allowed to mount the bitch. Being just to the side of the stud will allow for movement of the stud and still remain close enough to use the artificial vagina for the collection. It is important to have enough room to allow the stud to mount and then dismount during the collection.
Allow the male to mount. As he begins to thrust, gently massage the preputial sheath with one hand. If the male shows little interest in the bitch and does not mount, you may massage the sheath to stimulate an erection.
As erection occurs, pull the sheath behind the bulbus glandis. At the same time, with the other hand, slip the AV over the penis to just below the bulbus glandis.
Be careful when reflecting the sheath that the male does not have the bulbus glandis engorged. It is very painful if not impossible to reflect the sheath after the bulbus glandis has engorged. If needed the male may be removed from the room and walked until the bulbus glandis has gone down and then attempt the collection again. In some very aggressive studs it may be necessary to collect the sample with the sheath partially covering the bulbus glandis.
Reposition your hands providing gentle but constant pressure just proximal to and incorporating the bulbus glandis. You may stimulate the penis caudal to the bulbus glandis with your other hand.
Most dogs will thrust initially, coinciding with the penetration. They achieve full erection coinciding with the "tie", and will try to step over the bitch and your arm. You may help the stud by lifting his leg over your arm and turning his penis 180 degrees, so it is directed backwards between his legs.
You should be able to visualize the semen as it flows into the clear tube. Most dogs ejaculate in 3 fractions: an initial clear or slightly cloudy pre-ejaculate fraction, the sperm-rich fraction (SRF), and a clear fraction of prostatic fluid. The initial clear fraction is released during the period of vigorous thrusting. The SRF should appear as a thick, white, creamy liquid, which is usually released just as vigorous thrusting stops and the stud steps over. In some studs the SRF maybe thin white and the volume may vary from ½ cc to 10 cc. The clear prostatic fluid follows in varying amounts. When the SRF has been released and the first prostatic fluid is noted the collection is stopped. (If a teaser bitch is not used, then vigorous thrusting may not be observed.)
Switch tubes attached to the artificial vagina during the collection to separate the fractions. Because I centrifuge all samples I will not separate pre-ejaculate from SRF until the centrifugation process. If no centrifuge is available it is important to separate the fractions. After microscopic evaluation, the contents of tubes changed too early may be combined, if appropriate. Make sure you have collected the entire SRF before stopping. It is not necessary to collect the entire third fraction; save just enough to evaluate for abnormal cells, bacteria, etc.
It is very important to make sure the erection has subsided after the collection. In the winter frostbite is possible and the male is never allowed outside on cold days when the erection is present. Walking the stud after the collection will decrease the time needed to lose the erection. If the penis appears dry, you may apply a sterile lubricant at this stage. Make sure the penis is fully retracted into the sheath before putting the dog into a cage or with other dogs.
CHF: Once semen is collected how can it be stored chilled until use?
Shultz: If the fresh semen is not to be used for several days it may be centrifuged with Semen Separating Solution (Zoetis) and extended with Fresh Express Extender (Zoetis). The extended semen is labeled and put in a beaker with water and placed in a 40 degree farenheight refrigerator. The purpose of the water bath is to keep the sample from rapid warming and chilling if the refrigerator is opened. Our refrigerator is at a constant 40 degrees while most refrigerators turn on and off with a 5 to 8 degree temperature variation. Using this method we have successfully stored semen over 3 weeks for a breeding. The semen is checked every 2 to 3 days and the extender is changed as necessary to keep motility at the best level possible.
CHF: Let’s turn our attention to the bitch. What are the stages of the estrus cycle and the major hormones that regulate the cycle? Can you describe them to breeders?
Proestrus: This is the initial stage of the heat cycle. Proestrus will last 8 to 11 days in most bitches. Vaginal bleeding starts during this period. Estrogen is being secreted and this causes vaginal dilation, thickening of the vaginal walls to prepare for breeding and causes the initial attraction of males. During this stage the eggs are developing on the ovaries. The eggs are in a fluid sac called a follicle. As the bitch nears estrus these develop and are about ½ centimeter in diameter. We have examined ovaries at this stage and, in many cases, we can determine how many eggs the bitch will release at estrus.
Estrus: This is the breeding portion of the cycle. This is also called Standing Heat. This stage lasts 3 to 6 days. The bitch is now receptive to males and will stand and flag for the males. During estrus the bitch has a rapid drop in estrogen and a rise in progesterone. Progesterone is secreted by the ovaries at the sites of the follicles. The follicles rupture when LH (leuteotrophic hormone) is secreted by the pituitary gland.
Diestrus: When standing heat ends all canines go into a diestrus phase. Progesterone is secreted by the ovaries for the term of the pregnancy. The progesterone is secreted whether or not the bitch is pregnant. This is the reason human tests do not work in dogs. Humans only secrete progesterone if pregnant. Relaxin is a hormone that is released by the placenta and uterus interface. This hormone increases rapidly and may be evaluated as a pregnancy test in dogs. The test is available by Zoetis and may be used 20 days after breeding but best accuracy is at 30 days.
Anestrus: this is the recovery phase. Anestrus has very little hormone function present. In this stage the uterus is recovering and usually lasts 4 months.
CHF: What is ovulation timing and when is it advisable to use ovulation timing?
Many diagnostic and ancillary aids are available to assist in the timing of ovulation and the subsequent inseminations in the bitch. No single test or assay is fully reliable or completely correlates with the bitch’s exact stage of estrus. A single exam, vaginal smear, assay result, etc., provides very limited information. Ovulation timing is more accurate and breeding management more successful when multiple parameters are repeatedly evaluated. Useful aids utilized in practice to time breedings include:
Vaginal cytology is the evaluation of the surface cells of the vaginal tract. During the heat cycle the lining of the vagina thickens dramatically to prevent damage to the tract during breeding. Breeding only occurs during estrus and dogs do not need the thickened walls at any other time. The surface cells change from small cells that are round with a large nucleus called parabasals to large cells that may appear as cornflakes called superficial cornified cells.
Years ago timing with vaginal swabs was all that was possible for breeding but now we have many more accurate testing available to pinpoint the breeding day or days.
Vaginal cytology is done with a q-tip that may be moistened with saline. The swab is inserted in the vaginal vault and passed dorsally. It is then rubbed on the vaginal wall and removed. The swab is rolled on a slide to transfer the cells. The slide is then stained for examination of the cells. This is a good method to determine the stage of the cycle but it is a poor method to determine when to breed the bitch.
The vaginal cells change from parabasals cells to intermediate cells to superficial cells. The superficial cells then cornify – this is the corn flake appearance when the outer cell wall becomes irregular, the nucleus shrinks or is absent and little holes will appear in the body of the cell. In most bitches cornification happens at the estrus stage but in some bitches the cornification may take place very early in the cycle and remain cornified for many days.
Vaginal cytology is very accurate in determining the first day of diestrus or the end of the heat cycle. Within 24 hours the cytology changes from mostly cornified cells to a mixture of parabasals, intermediates, cornified cells and the white blood cell is also present.
Vaginoscopy is a procedure in with the cranial vaginal area is examined with a type of endoscope. The surface of the vagina changes during the heat cycle and goes form a smooth shiny surface to a thickened tissue with deep folds and a surface with many small wrinkles. Because we have very accurate progesterone timing this is rarely used today.
Luteinizing Hormone Assay:
This is a simple counter top assay from Zoetis. The most accurate parameter used in ovulation timing is the actual identification of the LH surge by direct measurement of canine LH levels. This method is recommended to arrive at a more precise estimation of the fertile period.
CHF: How accurate are these tests?
Shultz: We are aware that no test will pinpoint ovulation with 100% accuracy. Utilize as many diagnostic tools as possible. A single vaginal smear, a single LH or progesterone value, or a single vaginoscopic exam provides very limited information.
CHF: Finally, when should insemination be performed?
Shultz: Once the LH surge has been identified, count forward to determine the fertile period. The day of the LH surge is designated as day zero. Days 4-7 after the LH surge encompass the true fertile period, with peak fertility on days 5 and 6. We do vaginal or Transcervical breedings on days 3 and 5 after the Progesterone rise or LH surge, with frozen semen breedings on day 5. Breedings dates may vary with the repro vet being used for the breeding. The probability of successful fertilization is optimized by properly planning inseminations.
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