Chapter 32 ARTIFICIAL INSEMINATION, FRESH EXTENDED SEMEN, AND FROZEN SEMEN
The manual collection and subsequent deposition of semen into the vaginal vault of a bitch in estrus (standing heat) is a common procedure used by breeders and veterinarians. As a result, artificial insemination (AI) is frequently requested by the dog owner or handler. Fresh undiluted semen, semen mixed with an extender, or frozen semen is used in dogs. The chances of success are enhanced if the veterinarian has a good understanding of the estrus cycle, semen collection, and AI techniques, as well as potential pitfalls.
There are several situations in which AI may be used. The most obvious reason for AI is a perceived inability of the male and female to breed. For the bitch, this situation may involve problems such as vaginal strictures, conformational defects, rear leg weakness, psychological problems, and pain. AI is also used to ensure insemination early in estrus in a bitch with infertility of unknown cause. For the male, weakness, arthritis, back pain, premature ejaculation, and conformational defects that prevent intromission or a “tie” may prevent natural mating. AI may also be chosen because of a major size difference between the mates. Psychological problems (i.e., shyness, inexperience, dislike of the partner, previous breeding difficulties) may also result in the need for AI.
Some owners wish to use AI to avoid any possible venereal contact between their dog and its mate, thereby controlling the spread of potentially infectious diseases. This is not sound reasoning when the concern is the transmission of infectious agents from the dog to the bitch. Inseminating semen into the vagina still provides intimate contact between bitch and stud dog. Any infectious agent that could be transmitted from the dog to the bitch during natural mating also has the potential to be transmitted during AI. However, AI does avoid transmission of infectious agents from the bitch to the stud dog. All breeding dogs and bitches should be free of Brucella infection, as determined by appropriate tests (see Chapter 26).
Some male dogs (particularly Doberman Pinschers) experience prostatic bleeding and hemospermia after exposure to a bitch “in heat” (estrus; see page 1000). The bleeding may be associated with von Willebrand’s disease (VWD) but has also been observed in dogs not afflicted with VWD. Regardless, AI can be performed without any contact between stud dog and bitch, occasionally avoiding this problem.
Fresh extended and frozen forms of semen are being used with increasing frequency. Because semen collection is the difficult task, insemination of extended or previously frozen semen remains a relatively simple procedure. The shipment and use of fresh extended or frozen semen helps defray the cost and removes the hazards associated with shipping the female.
Semen is usually collected in an artificial vagina. We cut off and use one end of a sterilized, soft rubber, cone-shaped bovine artificial vagina. To this is attached a 12 to 15 ml plastic tube (Fig. 32-1; also see Fig. 27-3, page 934). The wide-mouth end of the cone is folded over and sealed with rubber cement to make a smooth, nonabrasive edge. A small amount of nonspermicidal lubricating jelly is applied to the inner surface of the rubber cone. This is the only surface that makes penile contact.
FIGURE 32-1 Artificial insemination equipment: sterile gloves, sterile soft rubber artificial vagina, sterile plastic tube that fits into the rubber vagina, and soft flexible plastic catheter for insemination.
The most difficult task in AI is stimulating the male to ejaculate. Once this has been accomplished, the balance of the procedure is quite simple. With most stud dogs, semen can be collected in a clean, quiet room with a nonslippery floor. A bitch is not often needed. However, for experienced males accustomed to natural breeding, a bitch in heat makes collecting semen easier and may improve the quality of the semen sample collected. Application of a topical pheromone preparation (Eau d’Estrus; Synbiotics, Malvern, PA) to a nonestrual bitch may also help. With the owner holding the stud dog to minimize movement and to protect the collector, the penis and bulbus glandis are gently massaged within the penile sheath. When the bulbus glandis begins to enlarge, the sheath is slipped posteriorly, and the penis with the bulbus glandis is exteriorized (Fig. 32-2). Failure to exteriorize both the penis and the bulbus glandis from the sheath usually results in an incomplete erection and failure to ejaculate or incomplete ejaculation, presumably due to pain.
Once the penis and bulbus glandis have been extruded from the sheath, the collector firmly holds onto the base of the penis, proximal to the bulbus glandis (Fig. 32-3). The thumb and index finger are used, providing both massaging movements and downward pressure around the base of the bulbus glandis. During or immediately after an erection has been achieved, aggressive pelvic thrusting movements by the stud dog, which accompany the onset of ejaculation, may make it difficult to place the artificial vagina over the penis. Fortunately, pelvic thrusting is typically short-lived (5 to 30 seconds), the initial phase of the ejaculate consists primarily of sperm-free prostatic fluid, and the sperm-rich second fraction of the ejaculate usually begins as the pelvic thrusting begins to subside. Therefore there should be no alarm over failure to collect ejaculate produced at the beginning of pelvic thrusting. Immediately after pelvic thrusting stops, many males try to “step over” the collector’s arm, as if dismounting the bitch. The collector should simply allow this movement by the male, which results in a 180-degree rotation of the engorged penis such that it is protruding caudally between the rear legs (Fig. 32-4). Digital pressure should be maintained on the bulbus glandis and collection continued until the ejaculate becomes clear (see Fig. 27-4, page 934).
FIGURE 32-4 After completion of vigorous pelvic thrusting, the dog typically steps over the collector’s arm, resulting in a 180-degree rotation of the penis such that the penis is now positioned caudally. Notice the collector’s thumb and index finger, which continue to apply pressure to the proximal part of the engorged bulbus glandis to maintain the erection (arrow).
(Courtesy A. Davidson, Davis, CA.)
Semen is usually collected for 2 to 5 minutes. This represents the typical duration of the second (sperm-rich) phase of ejaculation. The clear plastic tube should already have been connected to the rubber artificial vagina, and the apparatus can be held under the collector’s arm during the initial stimulation period to provide some warmth. Canine semen is relatively resistant to cold shock, which alleviates the need for warm water baths or incubators for holding semen. Use of a clear plastic collection tube allows visualization of the semen. One hand is kept over the plastic to avoid excessive light exposure. As long as the ejaculate is obviously whitish or creamy and cloudy (normal and sperm rich), the ejaculate continues to be collected. When the ejaculate becomes clear (sperm free), collection can be discontinued. If the collector is not certain when the male has stopped ejaculating the sperm-rich fraction, collection should be stopped after 5 minutes. Continued collection only dilutes the sperm with the third-phase clear, sperm-free prostatic fluid, resulting in cumbersome fluid volumes.
Collection of the third fraction of the ejaculate is not necessary for successful insemination. Enough of the third fraction should be collected to yield a final semen volume that is adequate for insemination. What constitutes adequate semen volume depends on the size of the bitch to be inseminated. The larger the bitch, the more semen is required for insemination. Adequate semen volume ranges from 2 to 10 ml, with bitches under 10 kg usually requiring 3 ml of semen or less and bitches over 20 kg requiring 5 ml of semen or more. The collection system containing sperm-rich semen can be exchanged for a clean system if there is any need to evaluate the prostatic fluid.
The bitch should be inseminated within 5 to 10 minutes of collection of the semen. After collection, the male can be taken out of the room to avoid distractions. Prior to insemination, the color and consistency of the semen should be noted and a small drop placed on a warm glass slide. This semen can be quickly evaluated microscopically to ensure that a normal number of live, progressively motile spermatozoa are present. During this time, the semen is kept warm by holding the tube in one’s hand, which also minimizes exposure of semen to potentially harmful ultraviolet rays. Alternatively, a drop of semen can be evaluated by a technician while the remainder of the semen is placed in the insemination device. Abnormal color or consistency, oligospermia, or dead sperm should be identified before insemination, because the breeder may wish to use a different stud dog. If these problems are not recognized until after insemination, another stud dog cannot be used if the litter is to be registered with the American Kennel Club (AKC).
Vaginal and uterine insemination techniques have been described for the bitch. For vaginal insemination, semen is deposited into the cranial vagina near the cervix with a long insemination pipette. This technique is typically done with insemination of fresh and chilled semen. For uterine insemination, semen is deposited directly into the uterus. This technique results in a higher pregnancy rate and larger litter size than vaginal insemination, presumably because a greater percentage of inseminated spermatozoa reach the uterotubal junction (Thomassen et al, 2000). Uterine insemination techniques require specialized equipment or surgery. Uterine insemination is recommended with frozen-thawed semen because of the decreased viability of previously frozen spermatozoa.
Although a variety of “tools” are available for vaginal insemination, we routinely use a 12 ml syringe, flexible disposable male urinary catheter or rigid plastic insemination pipette (Synbiotics; Malvern, PA), and surgical gloves. The catheter or insemination pipette should be long enough to reach the cranial vagina, which can be estimated as half the length from the vulvar lips to the costal arch (Root Kustritz and Johnston, 2000). These items should be sterile. After the gloves have been put on, the semen sample is drawn into the syringe, the sterile catheter is attached, and the syringe is filled with an additional 1 to 3 ml of air. The bitch’s rear end is elevated above her head during and immediately after completion of the insemination. Usually we have owners sit in a chair and place a drape over the lap, and then hold the rear legs of the bitch in the lap (Fig. 32-5). With a technician holding the tail to the side, a gloved, nonlubricated index finger (except in very small dogs) is placed into the vaginal vault, palm up. If a lubricant is used, it must be nonspermicidal. The catheter is slid over the top of the finger and passed into the vaginal vault, avoiding accidental catheterization of the urethra. Sliding the catheter over the index finger also aids in avoiding the clitoral fossa. The catheter follows the dorsal curvature of the vaginal vault. The catheter is inserted until resistance is met. The resistance indicates that the cranial end of the vaginal vault has been reached or that the catheter has simply become trapped within vaginal folds. The catheter should be gently advanced as far cranially as possible before the semen is deposited to ensure deposition of spermatozoa near the cervix. Blind cannulation of the cervix is difficult because of its relative inaccessibility and anatomy (Roszel, 1992).
FIGURE 32-5 Owner sitting with rear quarters of bitch in her lap during insemination process to allow gravity to help move semen into uterus. The bitch is maintained in this position for 20 minutes after insemination.
When the syringe containing the semen has been emptied, it should be disconnected from the catheter, filled with a few more milliliters of air, reattached to the catheter, and emptied, thereby depositing any semen that may have remained in the catheter. Care should be taken to avoid injecting too much air into the vagina, as this may result in loss of semen out of the vulva once the procedure is complete.
Once the semen has been deposited, the catheter should be removed; the bitch’s hindquarters should remain elevated above her head for a minimum of 20 minutes, aiding the movement of semen anteriorly in the reproductive tract and into the uterus. Insertion of a gloved finger and gentle stroking of the dorsal wall of the vagina (i.e., “feathering”) during this time may stimulate muscular contractions within the reproductive tract, enhancing the movement of spermatozoa toward the ovaries. Unfortunately, no studies have evaluated the effectiveness or need for this procedure.
After the period for elevation of the hindquarters has been completed, the bitch should be kept quiet for an hour or so to minimize loss of semen out of the vagina. In addition, pressure should not be applied to the abdomen (e.g., lifting the dog up by the abdomen).
The only material reused is the rubber cone artificial vagina. Although gas-sterilized equipment is claimed to harm sperm, we have seen no problem with reused rubber cones. Perhaps this is because the cone is used primarily as a funnel and the semen is retained in the clear, new, disposable plastic tube.