Key Points

• Artificial insemination requires knowledge of the endocrinology of the estrous cycle in the bitch.
• Each method of insemination and form of semen preparation has advantages and disadvantages.
• Ovulation and insemination must be coordinated for a successful outcome.

Artificial insemination is an imitation of the natural act of mating. It involves instillation of semen removed from a male dog into the cranial vagina or uterus of a bitch during the most fertile time of her estrous cycle. It is most frequently indicated in bitches that have normal estrous cycles but have a history of failure to conceive after natural mating or inability to be bred naturally. Successful artificial insemination results in pregnancy.

Artificial insemination is an assisted reproductive technique that can be used to compensate for some causes of canine infertility. Many reasons may lead to the request for artificial insemination in dogs. In most cases, inability or unwillingness to copulate naturally or difficulty in achieving or maintaining a successful pregnancy is involved in the decision to seek help.^1,2 Assisted reproduction in bitches that do not cycle or that have abnormal cycle patterns presents a different challenge and is not covered in this article.

Prebreeding Concerns

Physical Examination

Ensuring that both the dam and sire are healthy is important. The general health of the dam and sire is assessed with a breeding soundness and prebreeding examination, respectively.^2-5 Only healthy animals without heritable genetic defects should be considered for artificial insemination. Information concerning possible heritable diseases should be obtained by questioning the owner. A general physical examination of the dam should be performed before, not at the time of, insemination to identify any problems. Likewise, the sire's semen quality should be ascertained well in advance of the insemination procedure. Poor-quality semen may result in an unsuccessful pregnancy or contribute to small litter sizes.

Even with a healthy dam and sire, insemination may not be successful. The experience of the person performing the procedure and the difficulty of the technique used may influence the outcome. Coordination of ovulation in the bitch, semen acquisition from the desired stud dog, and insemination during the most fertile period of estrus is the most critical aspect of artificial insemination. Therefore, the owner should be made aware of every factor of artificial insemination before proceeding, including the time commitment and financial cost of the procedure. A checklist can be used as a guide to educate the owner. Although discouragement is not warranted, the owner must fully understand the process and be advised of the probability of success or failure.

Estrus and Ovulation

The estrous cycle in bitches is divided into four stages: proestrus, estrus, diestrus (or metestrus), and anestrus. Artificial insemination is always performed during estrus because it is the stage when ovulation occurs. This is the most fertile time of the estrous cycle.^1-3 Estrus can last from 3 to 21 days in bitches, but the average duration is 9 days.^3,6 External signs and the behavior of the bitch during proestrus and estrus are related to hormonal changes.

During proestrus, follicle-stimulating hormone secreted by the pituitary gland induces the development and growth of ovarian follicles. The ovarian follicles secrete estrogen, and as estrogen levels increase, the vulva begins to swell and the uterus releases a sanguineous discharge. The swelling of the vulva is caused by swelling and proliferation of the vaginal mucosa. These changes can be seen via endoscopy as an increase in the height of the longitudinal vaginal folds. The vaginal epithelial cell type also changes in preparation for copulation; detection of this cell type on a vaginal smear indicates an increase in estrogen. The male dog becomes interested in the bitch as a result of pheromones that are secreted at this time. The bitch may seem interested in the male dog but is not receptive to mounting and intromission.

In early estrus, the ovarian follicles continue to grow and secrete estrogen. As estrus progresses, the estrogen levels begin to decline as the luteinized follicular cells begin to produce progesterone. In late estrus, declining estrogen levels and rising progesterone levels trigger the secretion of a high level of luteinizing hormone (LH) from the pituitary gland. Ovulation occurs 24 to 48 hours after this peak increase in LH. At this time, the vulva is turgid and less swollen. The uterine discharge becomes serosanguineous and decreases or completely stops. The predominant cell type of the vaginal mucosa changes again, the vaginal mucosa becomes less edematous, and the bitch becomes receptive to the dog.

Released oocytes cannot be fertilized immediately after ovulation, when they are at the beginning of the uterine tube. The most fertile days are 2 to 3 days after ovulation,^3,6 when the oocytes have descended through most of the uterine tube and are ready and available for fertilization. Natural mating or artificial insemination can be instituted during this 2- to 3-day period.

After ovulation, the remaining follicular cells develop into corpora lutea (yellow bodies) that continue to secrete progesterone. This continued rise in progesterone signals the onset of diestrus. In diestrus, there is another change in the primary cell type of the vaginal mucosa and no vulvar discharge. Vaginal and vulvar swelling decrease, the vulva gradually decreases in size, and the bitch again becomes nonreceptive to the dog.

Methods of Ovulation Timing

Ovulation timing is the determination of the most fertile days of estrus. Ovulation timing is used as part of the artificial insemination procedure to ensure that ova are present in the uterine tube at the time of the semen injection. An advantage of ovulation timing is that it allows the time of whelping to be determined exactly, which is especially important when a cesarean section is needed.

Vaginal Cytology

Vaginal cytology is the laboratory test most commonly used to confirm proestrus and estrus. For this test, a microscope slide is prepared from vaginal smears made by inserting a sterile cotton swab through the vulva into the vagina, avoiding the vestibule. A speculum or finger may be used to guard the swab on entry into the vagina. The cells from the cranial vagina walls easily exfoliate upon swabbing. After the cells are collected, the swab is rolled onto a glass microscope slide. The slide is allowed to air dry and is then stained, usually with a Diff-Quik stain, although other stains can also be used.

In proestrus, vaginal epithelial cells change from small parabasal cells with large nuclei to larger intermediate cells and then to very large cornified cells. The parabasal cells have basophilic blue-staining cytoplasm. The nuclei are easily seen, and the amount of nuclear material is roughly equal to the amount of cytoplasm. Intermediate cells are larger than the parabasal cells and less basophilic. The nuclei are smaller in comparison to the cytoplasm, and the cell borders are still somewhat rounded. Cornified cells have small or nonexistent nuclei and are large and square. High numbers of intermediate and cornified epithelial cells, red and white blood cells, and bacteria are evident during proestrus.

In estrus, the number of cornified cells increases. At the time of ovulation, the primary epithelial cell type seen on vaginal cytology is the cornified epithelial cell. The microscopic visualization of primarily (usually 90%) superficial epithelial cells and abundant bacteria without white blood cells indicates late estrus and probably ovulation.

The accuracy of vaginal cytology depends on the experience of the person(s) performing the swab, preparing the slide, and reading the slide. Therefore, it may be beneficial to use additional methods of determining ovulation to confirm the results of vaginal cytology.

Vaginoscopy

The hormone-related changes in the vaginal mucosa that take place during proestrus and estrus can be easily seen with the proper equipment and are consistent enough to determine the time of ovulation.⁶ The vaginal mucosa can be examined using a flexible endoscope, a pediatric sigmoido­scope, or, in very small dogs, an otoscope. Rigid endoscopes (cystoscopes) are also available for this purpose and for the observation of transcervical insemination. Observation of the vaginal mucosa to determine ovulation timing is referred to as evaluation of vaginal mucosal crenulation.

The increase in estrogen during proestrus leads to vaginal mucosal edema. The mucosa appears swollen and smooth and makes visualization of the vaginal lumen difficult. As the estrogen levels decrease, so does the edema. This decrease in swelling leads to a wrinkling of the vaginal mucosa, called crenulation. Initial crenulation indicates the beginning of the LH peak as the estrogen levels drop. After ovulation, marked wrinkling is seen and the vaginal lumen appears open. The disadvantages of determining vaginal mucosal crenulation are the requirements of prior experience, additional equipment, and sedation.

Hormonal Assays

Progesterone Assays

Measurement of serum progesterone during the estrous cycle can also be used to determine ovulation. The use of serum progesterone levels to determine ovulation is based on a normal physiologic increase in progesterone from basal concentration levels (<1 ng/dl) to >2 ng/dl just before ovulation. At the time of ovulation, progesterone levels increase to >4 ng/dl; after ovulation, they are even higher (levels vary with number of follicles). Progesterone is secreted by the corpora lutea after ovulation and signals that the follicle has ruptured and released the egg (ovulation has occurred).

Progesterone levels are measured using a serum sample. The commercial laboratory usually performs a radio­immunoassay or chemiluminescence test to determine serum progesterone levels. It is important that the commercial laboratory measure canine progesterone levels because the assay is species specific. This testing is readily available to the practicing veterinarian; however, a disadvantage may be the turnaround time for obtaining results. Obtaining results over holidays and weekends also may be problematic. Choosing a laboratory that performs the assay daily, allows overnight delivery of samples, and is willing to phone, fax, or email results daily is recommended. Planning is important to ensure that the laboratory will be available when needed. Laboratories that perform this ser­vice may be readily found locally or online. Some offer chilled semen service along with progesterone assays.

In-house tests that measure canine progesterone are available. Most require a serum or plasma sample. All require refrigeration and have a shelf life of about 60 to 90 days. The manufacturer's directions must be followed carefully for each test. Samples must be prepared properly because hemolysis may cause a false reading in some tests. Lipemia may also cause false readings on some tests; before these tests are used, the animal may need to be fasted. The tests usually come with standard positive and negative samples for comparison. Results are given as a range rather than a specific number value. The available tests are the PreMate (Camelot Farms, College Station, TX), K9-Proges-Check (Endocrine Technologies, Newark, CA), Status Pro (Synbiotics, San Diego), and Target Canine Ovulation Testing Kit (BioMetallics, Princeton, NJ).

Luteinizing Hormone Assays

The accuracy of determining ovulation timing can be improved by adding an in-house LH assay to the progesterone assay. The LH assay measures the peak LH level that occurs before ovulation. Canine LH assays are available at some reference laboratories but are expensive and not widely used clinically. However, two in-house assays — the K-9 Ovicheck (Endocrine Technologies) and the Status-LH (Synbiotics) — are available. The K-9 Ovicheck assay is a quantitative assay marketed for research professionals to monitor physiologic and pathologic conditions related to circulating LH. The in-house use of this test is limited by its availability and complexity (it requires a two-step incubation process) and the time needed to complete the assay (3 hours). The Status-LH assay is a semiquantitative ELISA that is more readily available and less time consuming. However, because this test is semiquantitative, the results are either positive or negative. The results turn from negative to positive with an increase in serum LH levels consistent with the LH surge. Daily testing with this kit is required, and because preovulatory peaks can occur, an in-house pro­gesterone assay should also be used to ensure that the LH peak is associated with ovulation. Therefore, the major disadvantage of this assay is that it requires serial serum sampling and must be combined with the progesterone assay for accuracy. Ovulation occurs 2 to 3 days after the LH peak; at this point, the progesterone assay should be >4 ng/dl.

Semen Preparation

Fresh, chilled, or frozen semen may be used for artificial insemination. Semen is collected from male dogs using an artificial vagina and manual stimulation. Latex products should be avoided because latex has been reported to decrease sperm motility.⁷ Collected sperm should be analyzed for numbers, viability, motility, and morphology.^4,5 The conception rate is best with fresh semen (80%), followed by chilled (60%) and frozen (50% to 60%), but may vary according to the insemination technique used and the skill of the operator. The conception rate also depends on the proper handling of the semen and the fertility of the bitch.

Semen Analysis

Semen analysis, consisting of a sperm count to determine sperm concentration, motility analysis to determine sperm viability, and morphology analysis to identify sperm abnormalities, is performed to determine semen quality. Sperm concentration varies; therefore, sperm counts are conducted using a 1/100 Unopette dilutor (Becton Dickinson) and a hemocytometer. The diluted semen sample is used to charge the hemocytometer, which is placed under the microscope. The sperm present in the middle square of the nine large squares of the hemocytometer are counted and the number multiplied by 1 million to give the number of sperm cells per milliliter. This is multiplied by the volume of the ejaculate to determine the total number of sperm per ejaculation. Sperm counts of >200 million are usually seen in the rested stud dog. Counts of at least 200 million motile sperm are required for reliable vaginal artificial insemination.

Viability is determined by the amount of sperm with forward motility. Therefore, motility is considered the most important parameter in measuring semen quality. Motility can be analyzed by placing a drop of undiluted semen on a slide and placing it under the microscope for examination at 200 to 400x magnification. At least 70% of the sperm should exhibit forward motility.

Sperm morphology is analyzed to determine the percentage of morphologically normal sperm in the semen. Abnormal morphologic changes are divided into primary and secondary abnormalities. Primary abnormalities occur in the testes during spermatogenesis. Examples of primary abnormalities are ab­normal heads; double midpieces and/or tails; frayed, thickened, bent, kinked or ruptured midpieces; and coiled tails. Secondary abnormalities occur during transit through the epi­didymis, semen collection process, or preparation of the slide for evaluation. Examples of secondary abnormalities are detached normal heads, detached acrosomes, and bent tails.

Sperm morphology is evaluated by mixing a drop of semen with a drop of eosin-nigrosin stain. The mixture is carefully drawn slowly across the slide like a blood smear and allowed to air dry. Sperm morphology is observed at 1,000x magnification under oil immersion. Normal morphology should be >60% normal sperm that do not present with head, proximal droplet, or tail abnormalities. Primary and secondary defects should constitute <10% and <20%, respectively, of abnormalities seen. Morphologic abnormalities in more than 20% of sperm have been reported to lead to a decrease in litter size.

Fresh Semen

Fresh semen has a short life span without preservatives and an energy source. Fresh semen can be immediately instilled by artificial insemination methods if the female has already undergone ovulation timing.

Chilled Semen

Frozen or chilled semen can be used if the male dog is not at the same location as the bitch. To prepare chilled semen, fresh semen is centrifuged to make a pellet, which is resuspended in a commercial extender that serves as an energy source for the spermatozoa. The semen is slowly chilled for an hour to preserve the longevity of the sperm during transport. The method used for further preparation and packaging depends on the semen supplier. Chilled semen must be shipped overnight or as soon as possible (ideally in 24 hours) to the owner of the female dog that is to be bred. Testing to determine ovulation timing must be conducted before the semen is shipped.

Frozen Semen

Frozen semen is initially prepared in the same way as chilled semen. The extender for frozen semen contains glycerol to prevent damage to the sperm during the freezing procedure. After the semen has been chilled for an hour, it is placed in straws or pelleted and frozen using dry ice or liquid nitrogen, depending on the semen supplier. The frozen semen samples must be labeled with the stud dog's name or other identification and the date of freezing. Semen prepared in this manner can be shipped frozen for use at a later date; therefore, ovulation timing is not necessarily performed before the semen is shipped. Frozen semen must be kept at -4°F (-20°C) until needed and must be used immediately after being thawed. The sperm have decreased viability after the freezing process and must be placed directly into the uterus. Therefore, frozen semen cannot be used with intravaginal insemination.

Frozen and chilled semen should be prepared by an American Kennel Club (AKC)-recognized collection and storage facility for registration of the litter.⁸ There are 160 AKC-recognized collection and storage facilities in the United States. The names and locations of these facilities are available on the AKC Web site. It is important that the owner of the female dog to be bred keep in close contact with the semen supplier and veterinarian to coordinate artificial insemination at the most fertile time of estrus.

Methods of Artificial Insemination

Two methods of artificial insemination are described in the literature: intra­vag­inal and intrauterine. The intravaginal method involves instillation of semen into the vagina using a syringe and a long plastic (insemination) catheter. Insemination catheters for species other than dogs (e.g., goats, cows, horses) may be used. The catheter is introduced into the vulva and advanced as far as possible into the vagina. When the catheter cannot be advanced any further, a syringe containing the semen is attached. The bitch is held at a 45° angle with her hind feet in the air while the semen is injected. The syringe is removed, and the bitch remains with her hind end elevated for 15 to 20 minutes to allow gravity to assist sperm transportation to the uterus. A gloved finger may be inserted into the vagina to gently stroke (feather) the vaginal walls to try to stimulate vaginal contractions to assist in moving the sperm cranially toward the cervix. This procedure requires little experience and can be used for multiple serial inseminations daily or every other day. The main disadvantage is that frozen semen cannot be used for this procedure.

Transcervical insemination involves instillation of semen into the uterus through the cervix. There are two methods described for transcervical insemination. The Scandinavian or Norwegian approach requires the use of a specialized metal catheter with a nylon sheath. The special catheter and sheath are introduced through the vulva into the cervix, which has been located by abdominal palpation. When the operator is sure that the catheter and sheath are in the cervix, the sheath is removed. The other transcervical method, known as the New Zealand approach, involves mild sedation and a rigid endoscope (cystocope) with a special port that allows visualization of the cervix and placement of an 8-Fr polypropylene catheter into the cervix. Using either transcervical technique, the semen is injected via syringe into the uterus after placement of the catheter. The bitch is then handled similarly to the intravaginal approach. The advantage of the transcervical approach is that because the semen is placed into the uterus, fresh, chilled, or frozen semen can be used. It is also safe enough to be used daily or every other day because only mild sedation may be required. The main disadvantage of trans­cervical insemination is that both procedures require skill and special equipment.

Surgical intrauterine instillation of semen involves direct injection into the uterus by surgical laparotomy. This procedure requires general anesthesia, a small abdominal incision to exteriorize the uterus, and incision into the uterus to instill the fresh, chilled, or frozen semen. Its major advantage is that it allows the operator to directly observe the injection of the semen into the uterus. However, it requires anesthesia and surgery. Therefore, it is usually only performed once because repeated surgical procedures and anesthesia place the bitch and puppies at risk. Furthermore, this procedure may be banned in some countries as inhumane.

Role of the Technician

As an assistant to the veterinarian, the technician needs to know what tests may be conducted and which insemination procedure will be used as well as the supplies and equipment needed for each. In some cases, the technician is involved with the prebreeding or breeding soundness examination. The technician may also have the responsibility of preparing and possibly reading slides for vaginal cytology, collecting samples and conducting in-house LH and proges­terone assays, or collecting and mailing samples for commercial laboratory assays for progesterone and receiving the results. The technician may also be responsible for keeping up-to-date on what laboratory tests are available and which are preferred based on previous clinic experience.

Conclusion

Coordination is key to increasing the probability of obtaining a successful pregnancy through artificial insemination. Although time-consuming, well-coordinated efforts are more likely to be rewarded with success. Without thorough planning, artificial insemination may be a frustrating exercise. Preexamination of the proposed dam and sire is needed to determine infectious, metabolic, or genetic defects that could lead to potential spread of infectious disease, failure to conceive, or early termination of pregnancy. Ovulation timing should be used to determine when the bitch is most fertile, especially when shipping chilled semen or when using frozen semen, if only one insemination is possible. Knowledge of the semen preparation used is important in order to determine the appropriate insemination method.