Advanced Small Animal Reproductive Techniques

Introduction

The clinical practice of canine theriogenology is a rewarding subspecialty of small animal veterinary medicine. Although demanding of the clinician’s time and expertise, breeders tend to be loyal and compliant clients. A good reproductive practice generates its own referrals and is usually quite busy. Obstetrics and pediatrics are also rewarding parts of the specialty. Theriogenology incorporates the disciplines of reproductive physiology, endocrinology, embryology, genetics, metabolism, nutrition, critical care, anesthesia, pharmacology, and anatomy. The practice of theriogenology involves both medical and surgical procedures. Recently, new technologies and equipment have been developed to enhance the success of clinical reproductive practice. The purpose of this paper is to describe some of these techniques.

Transcervical Catheterization

Access to the uterus is hampered by the anatomy of the canine cranial vagina and cervix. Historically, in the United States, both intrauterine sampling and insemination were usually performed via laparotomy in the bitch. Laparotomy is often objectionable to clinicians and breeders, especially with respect to elective procedures such as artificial insemination, because it requires general anesthesia and is invasive. Additionally, the invasiveness of the procedure limits certain diagnostic tests (e.g., cytology, culture, and/or biopsy) and intrauterine insemination to a single event during each estrus. Obtaining a single diagnostic sample during estrus may not be optimal for providing a diagnosis when evaluating a bitch for apparent infertility at the time of breeding. Obtaining a uterine biopsy during anestrus may be more informative, but data are lacking. Furthermore, the impact of invasive diagnostics (i.e., those that require an incision in the uterine wall) on ovarian function and implantation is also unknown.

Laparoscopic approaches to the canine uterus have been used infrequently in the United States, as they require special equipment and expertise. With laparoscopy, diagnostic tests and insemination are also limited to a single event during estrus, because of the need for general anesthesia. Laparoscopy is less invasive than a laparotomy, but surgical incisions are still involved.

A transcervical approach to the canine uterus has always been desirable, but only recently has it become technically feasible. The cervix is relatively inaccessible from the vagina in the dog. A blind technique involving palpation of the cervix transabdominally and manipulation of a metal catheter into the cervical os has been advocated in the Scandinavian countries since the 1980s; but this technique has a steep learning curve, and there is potential for trauma to the genital tract if the procedure is performed incorrectly.¹ The vagina of the bitch is long, with the length from cervix to vulva (including the vestibule) reported as being 10 to 14 cm in an 11-kg bitch.¹ The cervix cannot be reached through the vagina for digital palpation in the bitch. The length of equipment necessary to visualize or approach the canine cervix is consequently long [Figure 1]. Additionally, the prominent vaginal mucosal folds of the estrual bitch and the position of the cervix in the cranial vagina require that the equipment be rigid to enable manipulation of the endoscope to the cervical os.

One method to place semen directly into the uterus that does not require surgery is the New Zealand technique of transcervical intrauterine insemination.¹ The equipment needed for this method of intrauterine insemination is more expensive than the cost of the Scandinavian method, but it is easier to learn and less likely to cause trauma to the bitch.² The New Zealand method utilizes a rigid cystourethrascope^a,b to visualize and catheterize the cervix. The equipment consists of a 36- to 41-cm × 3.5- to 4-mm (depending on the brand) cystourethroscope with a 30° viewing angle, a protective sheath that contains a channel for the catheter, ports for infusion or suction, a bridge containing an additional port for passage of the insemination catheter, a cold light source, and an optional camera and monitor [Figure 2]. An 8-French polypropylene urinary catheter is inserted into the channel within the sheath. This cystourethroscope can be used for insemination in all but the smallest bitches. In very small bitches, a shorter, rigid cystourethroscope with a 2.7-mm diameter can be used.

An estrual bitch is ideally placed on a specially designed, adjustable, hydraulic table that contains a large, flat restraining strap that can be placed gently around the abdomen [Figure 3]. The adjustable table should have a nonskid surface on which the bitch can stand, and the height of the table should be variable so the operator can work comfortably. Sedation is rarely required. The cystourethroscope is inserted into the vulva and vestibule and is directed dorsally to avoid the urethral papilla and clitoral fossa; then it is directed over the pelvic rim into the vagina [Figure 4]. The vaginal mucosal folds are viewed, and the cystourethroscope is directed cranially within the vaginal lumen. Air insufflation is not necessary when the dog is in estrus. The cystourethroscope is directed cranially until the dorsal median postcervical fold is visualized [Figure 5]. The dorsal median postcervical fold is recognized as a semicylindrical fold oriented longitudinally in the dorsal cranial vagina. The dorsal median postcervical fold is followed until the cervix is visualized as a small, reddish rosette or cauliflower-like structure on a tubercle [Figure 6]. The cervix can be visualized because the cystourethroscope has a viewing angle of 30° directed dorsally.

Once the cervix is visualized, an 8-French catheter is passed cranially into the vaginal cervical os and gently rotated through the cervical canal. Positioning the cystourethroscope so that it parallels the direction of the cervical canal (by lowering the operator end slightly) facilitates passage of the catheter into the uterus. The catheter should pass easily through the cervical os. If passage is difficult, the angle of the catheter can be adjusted slightly by turning the catheter or by further adjusting the angle of the scope. Once the catheter is inserted through the cervical os, semen is injected through the catheter [Figures 7A, 7B].

It is advisable that frozen semen not be thawed until the catheter has been successfully placed through the cervix, in case successful catheterization cannot be achieved or the process becomes prolonged. Attempting the technique on another day (if ovulation timing permits) or resorting to surgical intrauterine insemination may be necessary. Almost all breeds and sizes of dogs have been inseminated successfully using this technique. If the cervix cannot be catheterized and deep vaginal insemination is to be performed, it is best to withdraw the cystourethroscope and perform routine artificial insemination via a rigid infusion pipette, because semen will wick out along the sheath of the cystourethroscope and will not remain in the vagina.

The transcervical endoscopic technique has been reported as having pregnancy rates of 100% when insemination is done twice using 200 × 10⁶ cells per insemination.^3,4 Pregnancy rates of 85% may be achieved using as few as 30 to 50 × 10⁶ cells per insemination.^3,4 This type of insemination requires a substantial financial investment in equipment unless rigid endoscopy is already available in the practice. The investment is soon returned, however, as the approach is popular with breeders wishing to avoid anesthesia and surgery for intrauterine inseminations. Additional use of the equipment for diagnostic procedures, such as intrauterine cytology and culture and endometrial biopsy, improves its value.

The ease of transcervical catheterization may be recorded for each bitch using a numerical grading scale (e.g., from 1=very easy; taking ≤ 5 minutes to 5=not achievable). Repeat inseminations provide better coverage during the fertile period of the estrous cycle and usually improve success rates. Clinicians may want to consider offering transcervical insemination at a reduced fee to increase the caseload during their learning period. Successful catheterization of five to 10 bitches is usually enough experience to make the procedure relatively easy.

Concerns surrounding potential introduction of vaginal bacteria into the uterine lumen using transcervical insemination have not been realized thus far and are unlikely, given recent knowledge that normal vaginal flora can be found in the uterus during proestrus and estrus.⁵ Vaginal bacteria are introduced into the uterine lumen with natural breeding, and the uterus has an inherent ability to normalize its flora after estrus.⁵

Maintenance of the insemination equipment requires a 10-minute soaking of the immersible parts in dilute chlorhexiderm solution and thorough rinsing with distilled water. Stronger disinfectants should be avoided because of their spermicidal potential. The equipment does not have to be sterile for artificial insemination, as the vagina and semen are not sterile. The wide acceptance of transcervical insemination implies good conception rates in field situations. As with all reproductive procedures, success of the procedure is greatly influenced by the timing of ovulation in the bitch and the quality of semen from the male. An increased number of inseminations with fewer viable sperm per insemination may improve conception and is feasible using this technique.

Freezing of Semen

Although an exhaustive review of the different methods to prepare frozen semen is not included here, it is important to realize that a good-quality ejaculate must be collected before cryopreservation can be successful. Old or sick dogs with poor semen quality have semen that deteriorates after freezing and thawing. Preparation of semen for cryopreservation requires a method of standardizing the concentration of the sample; adding semen extenders that provide nutrients and buffers; using a cryoprotectant that protects the cells during the freezing process; and having a packaging system, a freezing method, and a storage facility. An ejaculate from a dog cannot simply be placed into a kitchen freezer and be expected to survive.

Many different semen extenders, cryoprotectants, concentrations of cryoprotectants, freezing methods, and freezing rates have been used.^6–11 One of the main differences between chilled semen extenders and frozen semen extenders is the addition of a cryoprotectant in the transport medium of the latter. Cryoprotectants help maintain cell integrity during the freezing and thawing process. The most common cryoprotectant used is glycerol. Cryoprotectants can adversely impact semen motility, so they are not included in chilled semen preparations.

A simple method of freezing canine semen that is currently used by a laboratory of one of the authors is described as follows.² After semen is collected, a complete analysis is performed. During the time the semen concentration is evaluated, the semen is diluted 1:1 with a commercially available semen refrigeration extender^c and centrifuged for 10 minutes at 900 × gravity. After centrifugation, the supernatant is removed and the pellet is resuspended to a concentration of 400 × 10⁶ cells/mL, using the same refrigeration extender. The sample is then placed in a 5°C refrigerator for 1 hour. An appropriate number of 0.5-mL semen straws are labeled with all the data required by the dog’s registry (e.g., name, breed, date, registration number, collection facility). After 1 hour, a commercial freezing extender containing 12% glycerol^d kept at 5°C is added to the cooled solution to make a final concentration of 200 × 10⁶ cells/mL in 6% glycerol. In a 5°C cold box, the 0.5-mL straws are filled by aspiration, and the ends are heat sealed [Figure 8]. The straws are then placed on a screen that is attached to a 3 cm-thick Styrofoam frame, which is floating in liquid nitrogen [Figure 9]. After 10 minutes, the straws are plunged into the liquid nitrogen. Fertility using semen frozen by this method has been anecdotally reported by practitioners as good.

The cryopreserved semen is stored at −196°C in commercially available liquid nitrogen tanks. Storage and inventory are important aspects after the semen is cryopreserved. Accurate records must be kept as to the location and number of straws that are frozen from each ejaculate. The semen storage tank must constantly be monitored to ensure that the −196°C is maintained. Storage tanks should be checked at least every 2 weeks to ensure that a minimum of 5 cm of liquid nitrogen remains in the tank. If frost is noticed on the tank, the tank may be damaged, and semen should be transferred immediately to a new tank.

Several commercial organizations offer proprietary semen-freezing franchises to veterinarians and technicians, and they also provide a centralized storage facility. Training on the proper techniques of semen freezing and thawing is included with the franchise, as well as advertising and a referral network.

Most dog breed associations are not as concerned about the quality of the frozen semen as they are about identification of the individual straws. The quality control of the final product is dependent upon the freezing facility. If every effort is made to collect good semen and to use good freezing practices, the quality of the frozen sample should be good. Since most veterinarians only deal with thawing semen that has been frozen by someone else, it is important to follow the instructions for thawing provided by each freezing facility. Thawing procedures for frozen semen vary widely. The author’s laboratory thaws straws at 50°C for 10 seconds; but some freezing facilities recommend thawing at lower temperatures for a longer time, and some recommend adding thawing media during the thawing process.^2,10 The freezing facility’s recommendations should always be followed when thawing cryopreserved semen. As with chilled semen breedings, the American Kennel Club requires proper paperwork to be completed as well as DNA identification of the stud (unless the dog was deceased before the requirement was instituted) before a litter can be registered using frozen semen.

In summary, attention must be paid to the quality of the ejaculate, handling of the ejaculate, and the insemination technique to attain acceptable conception rates using artificial insemination. If care is taken, conception rates using artificial insemination of frozen semen can be excellent. The optimum technique for insemination (i.e., one that combines the best conception rate and fecundity with the lowest sperm dose, is easy for the operator, affordable for the client, and atraumatic for the bitch) is not yet known; however, in the next few years, a standard semen dose and insemination technique will probably be widely adopted.

Obstetrical Monitoring

The standard approach to managing labor in the bitch and queen has involved the client observing the dam’s behavior, the progression of whelping/queening, and the physical condition of the neonates. Using this method, little accurate and timely information is made available regarding uterine activity or fetal viability. Telephone consultations between the veterinarian and breeder usually involve interpretation of subjective data, such as time between deliveries, color of the vaginal discharge, and presence of externally visible contractions. While the above data are generally acceptable for an uneventful delivery in a young, healthy dam, they are insufficient to manage difficult cases. Because many veterinarians are reluctant to encourage the expense and risk of an unnecessary cesarean section, and as higher-risk pregnancies and more valuable litters become common, more informative methods of monitoring the birth process are desirable.

The duration and ease of labor correlate closely with the number and vigor of live offspring born.¹² The uterus exhibits characteristic patterns of contractility (e.g., frequency and strength) during gestation and labor. During late-term pregnancy, the uterus may contract once or twice an hour before actual labor is initiated.¹³ The appropriate progression of active labor involves a gradual increase in the frequency and strength of contractions during the first stage, then a transition to frequent, strong contractions and relaxations (i.e., second stage) that result in the effective vaginal delivery of a neonate and the placenta (i.e., third stage). Uterine contractions vary in frequency from 0 to 12 per hour, and in strength from 15 to 40 mm Hg, with spikes up to 60 mm Hg.¹³ Contractions during active labor can be 2 to 5 minutes in duration.¹³

Abnormal, dysfunctional labor contractions may be weak or prolonged and may be associated with fetal distress. It is important to note that external abdominal efforts (“abdominal contractions”) are not indicative of the nature of concurrent uterine contractions. During effective labor, abdominal efforts enhance uterine contractions. During dystocia, uterine contractions may be absent or dysfunctional while the abdominal effort appears strong. Inappropriate uterine contractility early in labor can also result in placental disruption and loss of the neonates (“premature labor”).

The presence of fetal distress is reflected by deceleration of the fetal heart rates. The normal fetal heart rate at term is 170 to 230 beats per minute (bpm).¹⁴ Slowing of the heart rate associated with uterine contractions suggests mismatch of fetal and maternal (i.e., birth canal) size or fetal malposition, malpresentation, or malposture. Transient acceleration of the heart rate occurs with normal fetal movement. Fetal heart rates of ≤ 150 to 160 bpm indicate fetal stress.¹⁴ Fetuses with heart rates ≤ 130 bpm have poor survivability if not delivered within 2 to 3 hours, and fetuses with heart rates ≤ 100 bpm indicate the need for immediate intervention.¹⁴

A novel approach to canine and feline obstetrical monitoring involves the use of external monitoring devices to detect and record uterine activity and fetal heart rates [Figures 10A, 10B].⁵ These devices can be used in a home setting or in a veterinary clinic [Figures 11–13]. Interpretation of the contractile pattern strips produced by the uterine monitor requires training and experience. Commercially available monitoring devices currently transmit recorded information by modem to obstetrical personnel trained to interpret them. Subsequently, the attending veterinary clinician is notified if labor is not progressing normally. The company providing this equipment requires that individual veterinary orders be on file for each case.^e Recordings are made once or twice daily for an hour when prelabor home-monitoring is performed. Recordings are made intermittently over short periods during active labor or during monitoring in the veterinary clinic when dystocia is suspected. Sensors in the uterine monitor detect changes in intrauterine and amniotic pressures. The presence of normal prelabor uterine activity can also be detected. The onset of an organized pattern of uterine activity, with increased frequency and strength of contractions, heralds the beginning of stage 1 labor.

Because a prodromal drop in canine body temperature can be missed and does not always correlate well with the onset of first-stage labor, detection of early uterine contractions is very valuable and indicates the need to monitor the bitch closely, as whelping is imminent. In one study, 20% of bitches monitored for a drop in temperature had none detected, and only 38% experienced a demonstrable temperature drop within 36 hours of the onset of labor.¹⁵ The use of a uterine monitor also permits proactive identification of labor for planned cesarean sections when gestational length is not accurately known. The administration of oxytocin and calcium gluconate can be directed and tailored based on the results of uterine monitoring.

In bitches and queens with a history of delivery of immature, nonviable fetuses, for which no infectious or traumatic cause can be found, uterine monitoring can detect premature labor. Hypoluteiodism has been suggested as a cause of premature labor in the bitch and queen, but it may be a result of premature labor rather than the cause. Premature labor may initiate luteolysis through the action of prostaglandins released from the placenta and uterus. Administration of terbutaline to slow or stop uterine contractions may be effective if premature labor is diagnosed.

At normal term, absolute indications for cesarean section can be detected with monitoring before fetal death or maternal compromise occurs. The mortality rate of neonatal puppies (up to 7 days of age) reportedly declined from 33% to 6% with the use of uterine and fetal monitoring in one assistance dog colony.¹⁵ In addition, with the use of uterine and fetal monitors, the anxiety level of the owners is diminished, and the level of participation of the veterinarian in whelping or queening is improved. The cost to the client for monitoring is generally less than the price of one puppy or kitten, and client acceptance is generally excellent.

Conclusion

Novel equipment and new technologies have recently improved the reproductive services that veterinarians with interest and expertise in theriogenology can offer. Small animal clinical reproduction now approaches the advanced level of expertise achieved in the fields of zoological medicine and endangered species management. The creation of residencies and postdoctorate programs limited to small animal theriogenology will further promote developments in the field of companion animal reproduction.