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Examination of the Canine Genital System
Examination of the Canine Genital System
Ingemar Settergren, D.V.M., Ph.D.*
EXAMINATION OF THE BITCH HISTORY AND GENERAL ExAMINATION
The history is an important part of the examination. Reliable and detailed information facilitate and form the basis of the diagnosis. Important factors are the age and general health of the bitch, previous pregnancies, and number of viable and weaned pups. Date and nature of the last parturition and observations during the postpartum period have significance for future fertility. Information should be obtained about the fertility of related animals, previous medications and vaccinations, and feeding and management practices. The general examination should include breed, color, type of coat, body condition, and general demeanor. When necessary, temperature and pulse should be registered.
SPECIAL EXAMINATION
Inspection and Palpation of the Genital Organs
The external examination should not be overlooked as it may furnish valuable information for a diagnosis. The vulva should be inspected for conformation, size, and discharge if any. The normal vulva is relatively small and wrinkled, and the vulvar lips have a good tone. During estrus the vulva becomes swollen, and most of the wrinkles disappear. *Visiting Professor of Obstetrics and Gynecology, New York State Veterinary College. Dr. Settergren's current address is Royal Veterinary College, Stockholm.
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A swollen vulva in nonestrus animals may indicate hormonal disturbances or tumors in the genital organs. Tumors of the vulvar lips may be found in older bitches; squamous cell carcinoma forms firm ulcerated or necrotic lesions. Large vaginal tumors sometimes protrude from the vulva, and prolapse occurs in vaginal hyperplasia. A bloody discharge from the vulva normally is seen during proes- · trus and may persist through the later stages of heat, but it can also be the symptom of uterine or vaginal tumors. A purulent discharge, sometimes red or brownish red in color, is a sign of metritis or pyometra and is different from the normal brown, odorless lochia. Abdominal palpation is a valuable complement to radiography in diagnosing pyometra and ovarian and uterine tumors. The mammary glands should be examined for mastitis and tumors. In acute mastitis the gland is usually hot, swollen, and sometimes discolored. The secretion is purulent and varies in color and consistency. Aseptically collected samples should be examined for a causative agent. In mastitis one or several glands are enlarged, hard, and nodular on palpation. Affected glands may contain a mucopurulent or serous secretion. Vaginal Examination
Inspection. The inner part of the vestibulum and the vagina are inspected with an illuminated speculum of glass or metal. The color and appearance of the mucosa as well as the presence of secretion or exudate are observed. The normal anestrus mucosa shows longitudinal ridges and a pale color; during heat it is pinkish red and congested. The external cervical os is observed for color and for uterine discharge. Transmissible venereal tum9rs occur in the vagina as single or multiple firm masses with smooth or ulcerated surfaces. Cytologic examination. The microscopic examination of vaginal smears is a useful method to determine the stage of the estrus cycle in the bitch. The method can also be used for detection of pathologic conditions, e.g., disturbances of the normal cycle and uterine infections. To obtain material for the examination, a sterile cotton applicator or a glass rod is inserted well into the vagina and turned several times. The vaginal material is rolled directly on dry slides and stained according to Wright or Giemsa. For detailed studies of the nuclei and a more distinct differentiation between cornified and noncornified cells, a modification of Shorr's trichrome staining method has been used by Schutte (1967). Vaginal fluid also can be sampled by the use of a pipet. One drop of the fluid is mixed with one drop of 0.1 per cent aqueous toluidine blue on a clean slide and covered a few minutes later with a coverslip. The different phases of the estrus cycle are reflected in the vaginal
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smears, and the classification is based mainly on the appearance of the epithelial cells and the numbers of red and white blood cells (Fig. 1). Proestrus, which lasts about nine days, is characterized by a very high number of erythrocytes in the beginning of the period, and these cells may be found before the hemorrhagic discharge is observed. Leukocytes are found in the beginning of proestrus, but will gradually diminish toward the end of this phase. The epithelial cells undergo cornification during proestrus, so that toward the end of the period the number of nucleated cells is low and cornified cells predominate. During estrus, which usually lasts five to ten days, the vaginal smear is dominated by cornified epithelial cells. A low number of erythrocytes may still be present, whereas leukocytes completely disappear until after ovulation. Metestrus lasts two to three months. The leukocytes which reappear late in estrus increase considerably during the first ten days of metestrus, after which the number gradually decreases. The cornified epithelial cells are rapidly reduced in number and are replaced by nucleated cells. Amorphous debris derived mainly from disintegrated epithelial cells is seen during this period. Anestrus is characterized by large nucleated cells and a varying but usually low number of leukocytes. It is difficult to establish from a single smear the stage of the estrus cycle. For an accurate identification of the various phases of the cycle, smears should be examined every second or third day after the onset of bleeding. The Eosinophilic· Index (E. I.), which is based principally on the number of cornified epithelial cells compared to noncornified cells, has been suggested by Schutte ( 1967) as a useful method to establish the optimal time for mating, especially in bitches with aberrant cycles. Endometritis can be diagnosed from the vaginal smears and is indicated if the vaginal fluid contains an excessive number of leukocytes compared to what is normal for the stage of the cycle. Biochemical examination. The glucose content of the vaginal mucus is believed to increase at the time of ovulation and this can be demonstrated by commercially available impregnated paper strips which show color reaction after ovulation. The strips are inserted in the anterior vagina and left there for a minute before they are read. The results seem to vary, and the usefulness of the technique is doubtful. Microbiologic examination. In cases of abnormal discharge from the vulva, and after abortions or embryonic deaths, a microbiologic examination of vaginal fluids is valuable for confirmation of the diagnosis and for selecting treatment. After thorough cleaning of the vulvar region, a sterile speculum is introduced in the vagina. A sample of vaginal fluid can be obtained on a sterile swab or aspirated in a pipet.
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Figu re l.
Vaginal smears typical of d iffer e nt phases of th e estrus cycle of th e bitch .
A , Proestrus - Sh owin g presence of erythrocytes, s uper fic ial no ncornified epith elial
cells, mucin , a nd cell deb r is. B , Estrus - Cellular ele me nts are cornifie d and anuclear. Some cell d ebris is present.
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Figure I. C, Metestrus - E arly':stage still shows cornifi ed cells, but noncornified cells a re returnin g a nd leu kocytes are apparent (arrows). D, Anestrus - Pred ominantly noncornified superficial an d deep epithelial cells, some leukocytes, bacteria and debris.
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Hysterography
Radiographic examination can be used as an alternative to exploratory laparotomy in the diagnosis of pathologic changes of the uterus which involve little increase in size of the organ, e.g., endometrial hyperplasia, cystic hyperplasia, and myometrial cysts. The radiopaque solution is injected into the uterus through a metal catheter with the bitch under general anesthesia. Details of the procedure are described by Cobb (1959). Laparotomy
An exploratory laparotomy sometimes is necessary for examination of the genital organs or for confirmation of a tentative diagnosis. The ovaries can be inspected for the presence of cyst formations or tumors. The examination will also reveal adhesions in the ovarian bursa and malformations or obstructions of the oviducts which may cause a hydrosalpinx. The uterus is examined for malformations and for pathologic changes which are not visible with radiographic methods. In cases where embryonic death is suspected, residues for placental sites may be demonstrated between normally developed fetuses. The discrepancy between the number of corpora lutea in the ovaries and the number of remaining fetuses confirms the diagnosis of embryonic death. At the time of laparotomy, a sample for microbiologic examination can be aspirated from the uterus after puncturing the wall with a needle. Material for biopsy also can be obtained from the uterine wall or from tumors. Pregnancy Examination
Palpation. In the bitch pregnancy can be recognized by means of abdominal palpation approximately three weeks after conception unless the animal is nervous or very fat. At this stage the embryos are represented by distinct swellings well separated from each other and with a diameter of 12 to 15 mm. At four weeks the uterine distentions have become spherical and have reached a size of about 25 mm. This is the optimal time for early pregnancy diagnosis. From five weeks the fetal enlargements become confluent, and the uterine horns appear of uniform diameter, which makes the diagnosis difficult or impossible. During advanced stages of pregnancy it is often possible to palpate the individual fetuses. At this time one of the fetuses often can be palpated by rectal examination. The mammary glands start to grow from about midpregnancy; the teats become enlarged and feel turgid on palpation. During the last days of pregnancy a watery or serous secretion may be expressed from the teats.
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Radiography. After the end of the seventh week following conception the fetal skeletal structure will have become mineralized enough to produce a diagnostic radiograph. Sometimes the vertebrae and the ribs can be seen at six weeks. Radiography is of special value to demonstrate a single fetus which can be difficult to palpate. Ultrasonic method. Fetal circulation can be detected by an ultrasonic instrument which amplifies the reflected signals. If ultrasonic sound waves strike a moving object they are reflected at a slightly altered frequency and are converted into audible sounds. The reflected signals from blood flow in the fetal heart, the umbilical cord, and the uterine vessels produce characteristic sounds. Pregnancy can be diagnosed by ultrasonic sounds in bitches pregnant more than 30 to 35 days. A positive diagnosis always seems to be correct. The presence or absence of life in the fetuses at late stages of pregnancy also can be determined. No deleterious effects of the ultrasonic sounds have been found if the instrument is used according to instructions. Cytologic examination. Examination of vaginal smears has been suggested as a method of diagnosing pregnancy. Several slides should be obtained at one time. Lumps of large flat epithelial cells with elongated nuclei are typical of pregnancy. Smears from nonpregnant animals should be studied as controls. Infections may confuse the results.
Blood Examination The general morphologic and biochemical hematology which is important in cases of infectious processes of the genital organs will be dealt with elsewhere. There are, however, some special examinations of blood components which are important in the diagnosis of genital disorders in male and female dogs. Dogs infected with Brucella canis develop antibodies which can be demonstrated serologically. In bitches aborting about two weeks before term where puppies or fetal membranes cannot be recovered for examination, a tentative diagnosis is confirmed by the agglutination test. Males that have served infected bitches should always be tested, too. Chromosomal aberrations as causes for infertility have received much attention in human medicine during recent years and have been described in several domestic animals. The karyotype (systematic arrangement of the chromosomes of a single cell) of a dog can be studied in tissue cultures of leukocytes. The blood should be obtained aseptically and directly into a tube containing the tissue culture medium. It should be brought to the laboratory without delay. A relatively simple test for the genetic sex of an animal is the examination of smears from the buccal mucosa for sex chromatin or the demonstration of so called drumsticks in leukocytes. Details of the procedure are described by Barr ( 1965 ).
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EXAMINATION OF THE DOG HISTORY AND GENERAL EXAMINATION
The history and general examination of the dog should provide, where applicable, the same facts as mentioned earlier for the bitch. The breeding records are a very important part of the history for the male animal. The owner of the dog should provide information about the dates and results of all the matings, if possible, and at least those for the last year. The results should include the number of pups born and weaned. Special attention should be paid to cases of abortion or malformed pups. Information should be obtained about the mating behavior in the environment the dog is used to. Records of previous examinations of reproductive functions and medical treatments should also be studied. The reproductive performance in related animals is important, too. Inbreeding is known to cause impairment of sexual function.
SPECIAL ExAMINATION
Mating Behavior and Semen Collection
At natural mating the service is often preceded by some form of courtship. The dog is sexually stimulated by the smell of a bitch in heat and will smell and lick the vulvar region. Sight and hearing on the other hand are of less importance, as blind and deaf dogs will mate normally. The erection of the penis occurs in two stages. First, the arteries relax and the glans penis becomes filled with blood and partly erected. At this stage the dog mounts the bitch and introduces the penis into the vagina through a series of thrusting movements. The final erection develops after intromission, when the veins of the dorsal side of the penis become compressed partly by contractions of the muscles around the root of the penis, partly by the action of the vulvar sphincter muscles. Ejaculation in the dog can be divided into three relatively distinct phases. The first fraction of semen is ejaculated during the thrusting motions and consists of a clear watery fluid mostly from the glands of the urethral mucosa. The volume is 0.25 to 3 mi., and this fraction is emitted during the first 30 to 50 seconds of the ejaculation. The second fraction consists of 0.5 to 4 ml. of a white, more viscous fluid with a high concentration of sperm; it is produced during 50 to 80 seconds. The third fraction is again watery and clear, contains no or very few sperm, and lasts from 2 to 20 minutes. It is primarily prostatic fluid. The volume may vary from 1 to 25 mi. Semen for fertility control or artificial insemination should be collected in a quiet place on a floor which is not slippery; otherwise the
EXAMINATION OF THE CANINE GENITAL SYSTEM
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dog may feel uncomfortable and insecure. Preferably it should be done in the presence of a bitch in heat. Several different techniques are used for semen collection. The simplest and most commonly used method is digital manipulation. If a bitch in heat is present the dog is allowed to mount before semen is collected; otherwise, the penis is given gentle massage within the prepuce. When the erection starts, the prepuce is pushed back and pressure is applied with two fingers behind the bulb of the glans. By turning the penis backward between the hindlegs of the dog (Fig. 2) there will be an increased pressure on the dorsal part of the penis simulating the tie during natural mating. In this way it is also easier for the operator to observe the different phases of the ejaculation and to collect a clean sample. Semen can be collected in a test tube by means of a glass funnel, in a Pyrex glass beaker, or in a rubber cone connected to a test tube. The equipment should be rinsed in distilled water before sterilization. It should be warmed to body temperature before use to prevent cold shock to the semen. An artificial vagina can be used for collection, especially if semen is to be collected regularly over a long period. Harrop (1954) recommends an artificial vagina composed of an outer rubber casing with a length of 7 to 8 em. Inside this is a rubber liner, and between the outer case and the liner is a cylindrical rubber bladder connected to a valve through
Figure 2. Method of collecting semen from a dog.
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which air is pumped, allowing variation in the size of the lumen and also creating a pulsating pressure on the penis. When collecting semen, the penis is directed into the artificial vagina as soon as erection is obtained: either spontaneously in the presence of a bitch or after massage of the bulb of the penis. Pulsations of the liner provide further stimulation to ejaculation. The rubber of the inner liner and the cone should be tested for possible toxic effects on the semen before use. Silicon rubber is nontoxic to sperm, and an inner liner of plastic of the same type as for collection of ram semen may be used. Electroejaculation has been tried in dogs but requires general anesthesia. The method is not always successful and the semen may be contaminated with urine. Ejaculation induced by an electric vibrator of the same type as for home massage has been reported in men and dogs. A collecting cup consisting of a glass beaker with a small polyethylene funnel is attached to the rubber head of the vibrator and then applied to the glans of the penis. Ejaculation occurred in 65 per cent of the dogs on the first attempt, and the semen was of good quality. Semen Evaluation
If the dog has not ejaculated for a long time it is advisable to let him mate or to collect semen a few days before examination. This will improve the semen and make its evaluation more reliable. Volume and macroscopic appearance. The total volume of the ejaculate depends on the breed and size of the dog. In larger breeds, the total volume is up to 30 ml.; in miniature breeds it may only be a few milliliters. Normal whole semen has a grayish to ,white color, depending on sperm concentration and possible admixture of leukocytes or other foreign cells. In aspermia the semen is watery and clear, sometimes with a slight yellow color. Red or pink color of the semen occurs in dogs with prostatitis or balanoposthitis. Concentration and pH. The concentration can be fairly well estimated visually by an experienced examiner. For a more exact evaluation the number of sperm per cubic millimeter should be determined by counting in a hemacytometer or by measuring the light transmission in a photometer. The concentration of sperm per cubic millimeter in normal semen varies between 20,000 and 300,000, with an average of about 100,000. It may be higher if only part of the last fraction is collected. The total number of sperm per ejaculate is the best way to evaluate the sperm producing capacity of the dog. The number may be as high as 3,000 million, with a lower limit for normal ejaculate of about 100 million. The pH of normal whole semen is mostly between 6 and 7, depend-
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ing on the concentration. The sperm-rich fraction has the lowest pH and the third fraction the highest (Boucher et a!., 1958). Motility. Examination of motility should be done as soon as possible after collection, with the semen kept at a temperature of 30 to 37° C. A thin layer of semen under a coverslip is studied at 200 to 300 times magnification. The percentage of sperm with a forward movement is estimated. Normal dog semen shows good motility at 70 to 80 per cent; figures below 60 per cent are regarded as abnormal. Morphologic examination. The sperm morphology is mostly studied in stained slides under oil immersion. A simple staining method is to mix a drop of semen with a drop of India ink and prepare thin smears from the mixture. In more extensive studies and in diagnostic laboratories we recommend the staining method of Williams. Examination in a phase contrast microscope of semen preserved in buffered formalin is an excellent complement to the study of stained smears (Fig. 3). The deviations of the head morphology are not as frequent and decisive as in ruminants. However, a wide variation in the size of the sperm heads, especially a high number of small and pyknotic ones, is a sign of disturbance of spermatogenesis. Double heads or tails occur more often in dog semen than in the semen of other domestic animals (Fig. 3c). Abnormalities of the acrosome are not uncommon. The most common findings in semen of dogs with lowered fertility are abnormalities related to the tail, as bent and coiled tails and cytoplasmic droplets at the proximal end of the mid piece (unript; sperm). Supravital staining (live-dead) is a complement to the motility test and is based on the principle that dead or damaged sperm are penetrated by eosin. A small drop of semen is mixed with about five times the volume of a warm staining solution of eosin and a background stain (e.g., nigrosin) and smeared on warm slides. Two hundred sperm are counted and the number of unstained (living) cells expressed as a percentage of the total number counted. The occurrence of leuko~ytes and cells from the testes and the tubular part of the tract is studied in smears stained with hematoxylin and eosin. Leukocytes in the semen are relatively common in dogs, and usually are due to balanoposthitis. However, they also may come from the prostate or epididymis. Microbiologic examination of semen and urethral samples. In cases where the clinical findings or the history indicate infectious processes in the genital tract, a microbiologic examination should be undertaken. This should include bacterial identification and antibiotic sensitivity testing. After thorough washing and disinfection of the preputial opening and surrounding area, the penis is partly exposed through retraction of the prepuce, and the tip of the penis is washed with a disinfecting solution. The urethra is irrigated with 2 to 3 mi. of warm physiologic saline through a disposable plastic teat cannula. The penis is once more
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Figure 3.
Canine
semen.
A , Three normal sperm and one
coile d tail sperm in I n dia ink preparation. B , T ypical canine semen, ph ase contrast prepar ation . C. Four normal sperm, one double head. Phase contrast pre p ar ation.
washed, and a sterile silastic m edical tube with rounded ends is introduced in the urethra. The pelvic accessory glands are massaged via the rectum, and the expressed sample collected in a sterile tube. If the testes and epidid ymis seem to be affected , a semen sample can be collected under sterile conditions immediately afte r ward. Inspection and Palpation of the Genital Organs Scrotum, testes, and epididymis. Inspection of the scr otum reveals asymme try which may be caused by differences in the size of the testes,
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or by cryptorchidism. Enlargement of the scrotum may be due to increased size of the testes but may also be the result of inflammatory changes or neoplasms of the scrotal wall. A thickened scrotal wall often will disturb the thermoregulation and produce testicular degeneration by increased temperature. The spermatic cord should be checked for varicocele. Examination of the testes should evaluate their size and consistency and the possible presence of tumors. Markedly small testes are found in bilateral hypoplasia or as the result of severe testicular degeneration. Asymmetry may be due to unilateral hypoplasia or delayed descent of one testicle. Chronic orchitis results in a hard, nodular consistency of the testes. Testicular tumors are relatively common in old dogs. Some tumors produce estrogens and induce feminization. The epididymis is closely attached to the upper border of the testis. It should be examined for congenital aplasia, which can be unilateral or bilateral. Epididymitis is not uncommon in dogs, and may be caused by ascending nonspecific infections, by distemper, or by Brucella canis. Penis and prepuce. The prepuce is examined for development and inflammatory processes. An incomplete prepuce is sometimes seen in connection with intersexuality. A too narrow preputial opening leads to phimosis. Balanoposthitis is common in dogs and is characterized by a purulent discharge from the preputial opening. Transmissible venereal tumors may affect both the prepuce and the penis. They are single or multiple gray to red masses which often become ulcerated. Biopsy of the Testicles Testicular biopsy is used for studies of histology and karyotype. For taking a tissue sampl~ a small incision is made on the lateral side of the testis after local anesthesia. The tunica albuginea is incised between visible blood vessels. By slight compression of the testis a small piece of tissue will protrude above the surface of the testis. It is cut off with a sharp razor-scalpel and immediately placed in a fixative. For histologic studies Bouin's solution is used (not formalin). For study of the meiotic karyotype, the specimen is treated according to instructions for the examining laboratory. Examination of the Accessory Glands In dogs the normal prostate gland usually can be palpated through the rectal wall. It has two symmetrical lobes and the over-all measurements will normally not exceed 3 to 3.5 em. The hyperplastic gland is usually symmetrical and has a smooth surface. It may be displaced cranially and therefore difficult to reach. In prostatitis the gland is enlarged and the dog experiences pain at palpation. Irregular nodular enlargement is typical of carcinoma, and in this disease the gland often shows adhesion to surrounding tissues and extreme pain is produced by palpation.
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ARTIFICIAL INSEMINATION Although artificial insemination in dogs was first performed about 200 years ago, and an artificial vagina for dogs was developed in the beginning of this century, the method has not become common in breeding work. There are several reasons for this: methods for selection of genetically superior dogs have not been developed; and it is difficult to preserve and transport dog semen. Indications for artificial insemination are: 1. To bridge the distance between the dog and the bitch when they are kept far away from each other; 2. To increase the use of especially valuable dogs; 3. To achieve mating in animals with some traumatic injury which would prevent natural mating; and 4. To prevent the spread of genital infections.
Handling of the Semen The collection and evaluation of the semen have been described previously. If the bitch is present at the time of semen collection, whole semen is used for insemination immediately after collection. Whole semen can be kept for a few hours after cooling on a water bath and kept at 5 to 10° C. For storage beyond a few hours and for transport, the spermrich fraction of the ejaculate should be diluted 1:4 to 1 :8 with an extender. Several extenders have been used. One of the most successful has been heated skim milk. The milk is slowly heated to 92-94° C. (200° F.) and kept at this temperature for ten minutes. After cooling, antibiotics are added before use. Semen diluted in skim milk can be stored for 24 hours witho~t loss of fertility if sufficient sperm are inseminated. One pregnancy has been reported after storage of the semen for five days in this extender. Egg yolk buffered in different ways also has been used as an extender for dog semen, and has given good survival and fertility for at least 24 hours. Several attempts have been made to deepfreeze dog semen. Some authors report a good survival after freezing and storage for long periods. Motility is rapidly lost after thawing, however, and the frozen semen seems to be infertile as a rule. Only one experiment giving pregnancies with deep-frozen semen has been reported. In that experiment the semen was diluted in ll per cent lactose with 4 per cent glycerine and 20 per cent egg yolk and frozen in pellets (Saeger, 1969).
Insemination The correct time to inseminate is the day after the bitch has first accepted the male. The inseminations are repeated once or twice with intervals of 48 hours if the bitch is still showing good "standing" heat. In doubtful cases, especially in bitches with long heat periods, the vagi-
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nal cytology may be examined according to methods mentioned earlier to predict the optimal time for insemination. The bitch is held on a convenient table , and the vulva is cleaned carefully. A 5- or 10-ml. syringe containing the semen is attached to a 9-inch plastic inseminating pipet by a rubber connector. The pipet is inserted into the vagina as illustrated in Figure 4A. As it is advanced to the cervix the pipet is elevated and passed through the cervix if possible (Fig. 4B). The semen is gently injected and the pipet withdrawn. The rear quarters of the bitch are elevated for several minutes. Some authors insert a glove-covered finger into the vagina and "feather"
Figure 4.
Canine artificial insemination technique.
A , T he plastic pipette is inserted anterodorsally to the region of the cervix. B , Seme n is injected slowly and gently.
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Fi gu re 5. Finge r introduced into the vagina to simulate the normal "tie."
or stroke its dorsal wall during the interval the rear quarters are raised (Fig. 5). Conception rates approach those of normal mating if this technique is carefully followed. Preserved semen shipped long distances has not produced such high rates of conception.
REFERENCES l. Cobb, C. M. : Radiographic o utline of genital system of the bitch. Vet. Rec. , 71:66 , 1959. 2. Ford, L.: Canine cytology - an o verlooked clinical technic? Modern Vet. Pr act., 49:3 2, 1968. 3. G)er, H. T.: Estrous cycle in the bitch: Vaginal fluids . Veterinary Scope, 5 , 2 , 1960. 4. Harrop, A. E.: Reproduction in the dog. London, Bailliere, Tindall a nd Cox, 1960. 5. Helper, L. C .: Diagnosis of pregnancy in the bitch with an ultrasonic Doppler instrument. J. A. V. M. A. , 156.60, 1970. 6. Leon ard, E. P. : The Artificial Inse mination of Dogs. ln: The Artificial Insemination of Farm Animals, E . .J. Perry, ed. New Brunswick, Ru tgers University Press, 4th ed., 1968. 7. Saeger, S. W . .J. : Successful pregnancies utilizing frozen dog semen. Artif. Insem. Digest, 17: 12, 1969. 8. Schutte, A. P.: Canine vaginal cytology. J. Small Anim. Pract. , 8 :301, 1967.
Royal Veterinary College 10405 Stockholm Sweden
Ingemar Settergren, D.V.M., Ph.D.*
EXAMINATION OF THE BITCH HISTORY AND GENERAL ExAMINATION
The history is an important part of the examination. Reliable and detailed information facilitate and form the basis of the diagnosis. Important factors are the age and general health of the bitch, previous pregnancies, and number of viable and weaned pups. Date and nature of the last parturition and observations during the postpartum period have significance for future fertility. Information should be obtained about the fertility of related animals, previous medications and vaccinations, and feeding and management practices. The general examination should include breed, color, type of coat, body condition, and general demeanor. When necessary, temperature and pulse should be registered.
SPECIAL EXAMINATION
Inspection and Palpation of the Genital Organs
The external examination should not be overlooked as it may furnish valuable information for a diagnosis. The vulva should be inspected for conformation, size, and discharge if any. The normal vulva is relatively small and wrinkled, and the vulvar lips have a good tone. During estrus the vulva becomes swollen, and most of the wrinkles disappear. *Visiting Professor of Obstetrics and Gynecology, New York State Veterinary College. Dr. Settergren's current address is Royal Veterinary College, Stockholm.
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A swollen vulva in nonestrus animals may indicate hormonal disturbances or tumors in the genital organs. Tumors of the vulvar lips may be found in older bitches; squamous cell carcinoma forms firm ulcerated or necrotic lesions. Large vaginal tumors sometimes protrude from the vulva, and prolapse occurs in vaginal hyperplasia. A bloody discharge from the vulva normally is seen during proes- · trus and may persist through the later stages of heat, but it can also be the symptom of uterine or vaginal tumors. A purulent discharge, sometimes red or brownish red in color, is a sign of metritis or pyometra and is different from the normal brown, odorless lochia. Abdominal palpation is a valuable complement to radiography in diagnosing pyometra and ovarian and uterine tumors. The mammary glands should be examined for mastitis and tumors. In acute mastitis the gland is usually hot, swollen, and sometimes discolored. The secretion is purulent and varies in color and consistency. Aseptically collected samples should be examined for a causative agent. In mastitis one or several glands are enlarged, hard, and nodular on palpation. Affected glands may contain a mucopurulent or serous secretion. Vaginal Examination
Inspection. The inner part of the vestibulum and the vagina are inspected with an illuminated speculum of glass or metal. The color and appearance of the mucosa as well as the presence of secretion or exudate are observed. The normal anestrus mucosa shows longitudinal ridges and a pale color; during heat it is pinkish red and congested. The external cervical os is observed for color and for uterine discharge. Transmissible venereal tum9rs occur in the vagina as single or multiple firm masses with smooth or ulcerated surfaces. Cytologic examination. The microscopic examination of vaginal smears is a useful method to determine the stage of the estrus cycle in the bitch. The method can also be used for detection of pathologic conditions, e.g., disturbances of the normal cycle and uterine infections. To obtain material for the examination, a sterile cotton applicator or a glass rod is inserted well into the vagina and turned several times. The vaginal material is rolled directly on dry slides and stained according to Wright or Giemsa. For detailed studies of the nuclei and a more distinct differentiation between cornified and noncornified cells, a modification of Shorr's trichrome staining method has been used by Schutte (1967). Vaginal fluid also can be sampled by the use of a pipet. One drop of the fluid is mixed with one drop of 0.1 per cent aqueous toluidine blue on a clean slide and covered a few minutes later with a coverslip. The different phases of the estrus cycle are reflected in the vaginal
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smears, and the classification is based mainly on the appearance of the epithelial cells and the numbers of red and white blood cells (Fig. 1). Proestrus, which lasts about nine days, is characterized by a very high number of erythrocytes in the beginning of the period, and these cells may be found before the hemorrhagic discharge is observed. Leukocytes are found in the beginning of proestrus, but will gradually diminish toward the end of this phase. The epithelial cells undergo cornification during proestrus, so that toward the end of the period the number of nucleated cells is low and cornified cells predominate. During estrus, which usually lasts five to ten days, the vaginal smear is dominated by cornified epithelial cells. A low number of erythrocytes may still be present, whereas leukocytes completely disappear until after ovulation. Metestrus lasts two to three months. The leukocytes which reappear late in estrus increase considerably during the first ten days of metestrus, after which the number gradually decreases. The cornified epithelial cells are rapidly reduced in number and are replaced by nucleated cells. Amorphous debris derived mainly from disintegrated epithelial cells is seen during this period. Anestrus is characterized by large nucleated cells and a varying but usually low number of leukocytes. It is difficult to establish from a single smear the stage of the estrus cycle. For an accurate identification of the various phases of the cycle, smears should be examined every second or third day after the onset of bleeding. The Eosinophilic· Index (E. I.), which is based principally on the number of cornified epithelial cells compared to noncornified cells, has been suggested by Schutte ( 1967) as a useful method to establish the optimal time for mating, especially in bitches with aberrant cycles. Endometritis can be diagnosed from the vaginal smears and is indicated if the vaginal fluid contains an excessive number of leukocytes compared to what is normal for the stage of the cycle. Biochemical examination. The glucose content of the vaginal mucus is believed to increase at the time of ovulation and this can be demonstrated by commercially available impregnated paper strips which show color reaction after ovulation. The strips are inserted in the anterior vagina and left there for a minute before they are read. The results seem to vary, and the usefulness of the technique is doubtful. Microbiologic examination. In cases of abnormal discharge from the vulva, and after abortions or embryonic deaths, a microbiologic examination of vaginal fluids is valuable for confirmation of the diagnosis and for selecting treatment. After thorough cleaning of the vulvar region, a sterile speculum is introduced in the vagina. A sample of vaginal fluid can be obtained on a sterile swab or aspirated in a pipet.
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Figu re l.
Vaginal smears typical of d iffer e nt phases of th e estrus cycle of th e bitch .
A , Proestrus - Sh owin g presence of erythrocytes, s uper fic ial no ncornified epith elial
cells, mucin , a nd cell deb r is. B , Estrus - Cellular ele me nts are cornifie d and anuclear. Some cell d ebris is present.
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Figure I. C, Metestrus - E arly':stage still shows cornifi ed cells, but noncornified cells a re returnin g a nd leu kocytes are apparent (arrows). D, Anestrus - Pred ominantly noncornified superficial an d deep epithelial cells, some leukocytes, bacteria and debris.
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Hysterography
Radiographic examination can be used as an alternative to exploratory laparotomy in the diagnosis of pathologic changes of the uterus which involve little increase in size of the organ, e.g., endometrial hyperplasia, cystic hyperplasia, and myometrial cysts. The radiopaque solution is injected into the uterus through a metal catheter with the bitch under general anesthesia. Details of the procedure are described by Cobb (1959). Laparotomy
An exploratory laparotomy sometimes is necessary for examination of the genital organs or for confirmation of a tentative diagnosis. The ovaries can be inspected for the presence of cyst formations or tumors. The examination will also reveal adhesions in the ovarian bursa and malformations or obstructions of the oviducts which may cause a hydrosalpinx. The uterus is examined for malformations and for pathologic changes which are not visible with radiographic methods. In cases where embryonic death is suspected, residues for placental sites may be demonstrated between normally developed fetuses. The discrepancy between the number of corpora lutea in the ovaries and the number of remaining fetuses confirms the diagnosis of embryonic death. At the time of laparotomy, a sample for microbiologic examination can be aspirated from the uterus after puncturing the wall with a needle. Material for biopsy also can be obtained from the uterine wall or from tumors. Pregnancy Examination
Palpation. In the bitch pregnancy can be recognized by means of abdominal palpation approximately three weeks after conception unless the animal is nervous or very fat. At this stage the embryos are represented by distinct swellings well separated from each other and with a diameter of 12 to 15 mm. At four weeks the uterine distentions have become spherical and have reached a size of about 25 mm. This is the optimal time for early pregnancy diagnosis. From five weeks the fetal enlargements become confluent, and the uterine horns appear of uniform diameter, which makes the diagnosis difficult or impossible. During advanced stages of pregnancy it is often possible to palpate the individual fetuses. At this time one of the fetuses often can be palpated by rectal examination. The mammary glands start to grow from about midpregnancy; the teats become enlarged and feel turgid on palpation. During the last days of pregnancy a watery or serous secretion may be expressed from the teats.
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Radiography. After the end of the seventh week following conception the fetal skeletal structure will have become mineralized enough to produce a diagnostic radiograph. Sometimes the vertebrae and the ribs can be seen at six weeks. Radiography is of special value to demonstrate a single fetus which can be difficult to palpate. Ultrasonic method. Fetal circulation can be detected by an ultrasonic instrument which amplifies the reflected signals. If ultrasonic sound waves strike a moving object they are reflected at a slightly altered frequency and are converted into audible sounds. The reflected signals from blood flow in the fetal heart, the umbilical cord, and the uterine vessels produce characteristic sounds. Pregnancy can be diagnosed by ultrasonic sounds in bitches pregnant more than 30 to 35 days. A positive diagnosis always seems to be correct. The presence or absence of life in the fetuses at late stages of pregnancy also can be determined. No deleterious effects of the ultrasonic sounds have been found if the instrument is used according to instructions. Cytologic examination. Examination of vaginal smears has been suggested as a method of diagnosing pregnancy. Several slides should be obtained at one time. Lumps of large flat epithelial cells with elongated nuclei are typical of pregnancy. Smears from nonpregnant animals should be studied as controls. Infections may confuse the results.
Blood Examination The general morphologic and biochemical hematology which is important in cases of infectious processes of the genital organs will be dealt with elsewhere. There are, however, some special examinations of blood components which are important in the diagnosis of genital disorders in male and female dogs. Dogs infected with Brucella canis develop antibodies which can be demonstrated serologically. In bitches aborting about two weeks before term where puppies or fetal membranes cannot be recovered for examination, a tentative diagnosis is confirmed by the agglutination test. Males that have served infected bitches should always be tested, too. Chromosomal aberrations as causes for infertility have received much attention in human medicine during recent years and have been described in several domestic animals. The karyotype (systematic arrangement of the chromosomes of a single cell) of a dog can be studied in tissue cultures of leukocytes. The blood should be obtained aseptically and directly into a tube containing the tissue culture medium. It should be brought to the laboratory without delay. A relatively simple test for the genetic sex of an animal is the examination of smears from the buccal mucosa for sex chromatin or the demonstration of so called drumsticks in leukocytes. Details of the procedure are described by Barr ( 1965 ).
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EXAMINATION OF THE DOG HISTORY AND GENERAL EXAMINATION
The history and general examination of the dog should provide, where applicable, the same facts as mentioned earlier for the bitch. The breeding records are a very important part of the history for the male animal. The owner of the dog should provide information about the dates and results of all the matings, if possible, and at least those for the last year. The results should include the number of pups born and weaned. Special attention should be paid to cases of abortion or malformed pups. Information should be obtained about the mating behavior in the environment the dog is used to. Records of previous examinations of reproductive functions and medical treatments should also be studied. The reproductive performance in related animals is important, too. Inbreeding is known to cause impairment of sexual function.
SPECIAL ExAMINATION
Mating Behavior and Semen Collection
At natural mating the service is often preceded by some form of courtship. The dog is sexually stimulated by the smell of a bitch in heat and will smell and lick the vulvar region. Sight and hearing on the other hand are of less importance, as blind and deaf dogs will mate normally. The erection of the penis occurs in two stages. First, the arteries relax and the glans penis becomes filled with blood and partly erected. At this stage the dog mounts the bitch and introduces the penis into the vagina through a series of thrusting movements. The final erection develops after intromission, when the veins of the dorsal side of the penis become compressed partly by contractions of the muscles around the root of the penis, partly by the action of the vulvar sphincter muscles. Ejaculation in the dog can be divided into three relatively distinct phases. The first fraction of semen is ejaculated during the thrusting motions and consists of a clear watery fluid mostly from the glands of the urethral mucosa. The volume is 0.25 to 3 mi., and this fraction is emitted during the first 30 to 50 seconds of the ejaculation. The second fraction consists of 0.5 to 4 ml. of a white, more viscous fluid with a high concentration of sperm; it is produced during 50 to 80 seconds. The third fraction is again watery and clear, contains no or very few sperm, and lasts from 2 to 20 minutes. It is primarily prostatic fluid. The volume may vary from 1 to 25 mi. Semen for fertility control or artificial insemination should be collected in a quiet place on a floor which is not slippery; otherwise the
EXAMINATION OF THE CANINE GENITAL SYSTEM
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dog may feel uncomfortable and insecure. Preferably it should be done in the presence of a bitch in heat. Several different techniques are used for semen collection. The simplest and most commonly used method is digital manipulation. If a bitch in heat is present the dog is allowed to mount before semen is collected; otherwise, the penis is given gentle massage within the prepuce. When the erection starts, the prepuce is pushed back and pressure is applied with two fingers behind the bulb of the glans. By turning the penis backward between the hindlegs of the dog (Fig. 2) there will be an increased pressure on the dorsal part of the penis simulating the tie during natural mating. In this way it is also easier for the operator to observe the different phases of the ejaculation and to collect a clean sample. Semen can be collected in a test tube by means of a glass funnel, in a Pyrex glass beaker, or in a rubber cone connected to a test tube. The equipment should be rinsed in distilled water before sterilization. It should be warmed to body temperature before use to prevent cold shock to the semen. An artificial vagina can be used for collection, especially if semen is to be collected regularly over a long period. Harrop (1954) recommends an artificial vagina composed of an outer rubber casing with a length of 7 to 8 em. Inside this is a rubber liner, and between the outer case and the liner is a cylindrical rubber bladder connected to a valve through
Figure 2. Method of collecting semen from a dog.
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which air is pumped, allowing variation in the size of the lumen and also creating a pulsating pressure on the penis. When collecting semen, the penis is directed into the artificial vagina as soon as erection is obtained: either spontaneously in the presence of a bitch or after massage of the bulb of the penis. Pulsations of the liner provide further stimulation to ejaculation. The rubber of the inner liner and the cone should be tested for possible toxic effects on the semen before use. Silicon rubber is nontoxic to sperm, and an inner liner of plastic of the same type as for collection of ram semen may be used. Electroejaculation has been tried in dogs but requires general anesthesia. The method is not always successful and the semen may be contaminated with urine. Ejaculation induced by an electric vibrator of the same type as for home massage has been reported in men and dogs. A collecting cup consisting of a glass beaker with a small polyethylene funnel is attached to the rubber head of the vibrator and then applied to the glans of the penis. Ejaculation occurred in 65 per cent of the dogs on the first attempt, and the semen was of good quality. Semen Evaluation
If the dog has not ejaculated for a long time it is advisable to let him mate or to collect semen a few days before examination. This will improve the semen and make its evaluation more reliable. Volume and macroscopic appearance. The total volume of the ejaculate depends on the breed and size of the dog. In larger breeds, the total volume is up to 30 ml.; in miniature breeds it may only be a few milliliters. Normal whole semen has a grayish to ,white color, depending on sperm concentration and possible admixture of leukocytes or other foreign cells. In aspermia the semen is watery and clear, sometimes with a slight yellow color. Red or pink color of the semen occurs in dogs with prostatitis or balanoposthitis. Concentration and pH. The concentration can be fairly well estimated visually by an experienced examiner. For a more exact evaluation the number of sperm per cubic millimeter should be determined by counting in a hemacytometer or by measuring the light transmission in a photometer. The concentration of sperm per cubic millimeter in normal semen varies between 20,000 and 300,000, with an average of about 100,000. It may be higher if only part of the last fraction is collected. The total number of sperm per ejaculate is the best way to evaluate the sperm producing capacity of the dog. The number may be as high as 3,000 million, with a lower limit for normal ejaculate of about 100 million. The pH of normal whole semen is mostly between 6 and 7, depend-
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113
ing on the concentration. The sperm-rich fraction has the lowest pH and the third fraction the highest (Boucher et a!., 1958). Motility. Examination of motility should be done as soon as possible after collection, with the semen kept at a temperature of 30 to 37° C. A thin layer of semen under a coverslip is studied at 200 to 300 times magnification. The percentage of sperm with a forward movement is estimated. Normal dog semen shows good motility at 70 to 80 per cent; figures below 60 per cent are regarded as abnormal. Morphologic examination. The sperm morphology is mostly studied in stained slides under oil immersion. A simple staining method is to mix a drop of semen with a drop of India ink and prepare thin smears from the mixture. In more extensive studies and in diagnostic laboratories we recommend the staining method of Williams. Examination in a phase contrast microscope of semen preserved in buffered formalin is an excellent complement to the study of stained smears (Fig. 3). The deviations of the head morphology are not as frequent and decisive as in ruminants. However, a wide variation in the size of the sperm heads, especially a high number of small and pyknotic ones, is a sign of disturbance of spermatogenesis. Double heads or tails occur more often in dog semen than in the semen of other domestic animals (Fig. 3c). Abnormalities of the acrosome are not uncommon. The most common findings in semen of dogs with lowered fertility are abnormalities related to the tail, as bent and coiled tails and cytoplasmic droplets at the proximal end of the mid piece (unript; sperm). Supravital staining (live-dead) is a complement to the motility test and is based on the principle that dead or damaged sperm are penetrated by eosin. A small drop of semen is mixed with about five times the volume of a warm staining solution of eosin and a background stain (e.g., nigrosin) and smeared on warm slides. Two hundred sperm are counted and the number of unstained (living) cells expressed as a percentage of the total number counted. The occurrence of leuko~ytes and cells from the testes and the tubular part of the tract is studied in smears stained with hematoxylin and eosin. Leukocytes in the semen are relatively common in dogs, and usually are due to balanoposthitis. However, they also may come from the prostate or epididymis. Microbiologic examination of semen and urethral samples. In cases where the clinical findings or the history indicate infectious processes in the genital tract, a microbiologic examination should be undertaken. This should include bacterial identification and antibiotic sensitivity testing. After thorough washing and disinfection of the preputial opening and surrounding area, the penis is partly exposed through retraction of the prepuce, and the tip of the penis is washed with a disinfecting solution. The urethra is irrigated with 2 to 3 mi. of warm physiologic saline through a disposable plastic teat cannula. The penis is once more
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Figure 3.
Canine
semen.
A , Three normal sperm and one
coile d tail sperm in I n dia ink preparation. B , T ypical canine semen, ph ase contrast prepar ation . C. Four normal sperm, one double head. Phase contrast pre p ar ation.
washed, and a sterile silastic m edical tube with rounded ends is introduced in the urethra. The pelvic accessory glands are massaged via the rectum, and the expressed sample collected in a sterile tube. If the testes and epidid ymis seem to be affected , a semen sample can be collected under sterile conditions immediately afte r ward. Inspection and Palpation of the Genital Organs Scrotum, testes, and epididymis. Inspection of the scr otum reveals asymme try which may be caused by differences in the size of the testes,
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or by cryptorchidism. Enlargement of the scrotum may be due to increased size of the testes but may also be the result of inflammatory changes or neoplasms of the scrotal wall. A thickened scrotal wall often will disturb the thermoregulation and produce testicular degeneration by increased temperature. The spermatic cord should be checked for varicocele. Examination of the testes should evaluate their size and consistency and the possible presence of tumors. Markedly small testes are found in bilateral hypoplasia or as the result of severe testicular degeneration. Asymmetry may be due to unilateral hypoplasia or delayed descent of one testicle. Chronic orchitis results in a hard, nodular consistency of the testes. Testicular tumors are relatively common in old dogs. Some tumors produce estrogens and induce feminization. The epididymis is closely attached to the upper border of the testis. It should be examined for congenital aplasia, which can be unilateral or bilateral. Epididymitis is not uncommon in dogs, and may be caused by ascending nonspecific infections, by distemper, or by Brucella canis. Penis and prepuce. The prepuce is examined for development and inflammatory processes. An incomplete prepuce is sometimes seen in connection with intersexuality. A too narrow preputial opening leads to phimosis. Balanoposthitis is common in dogs and is characterized by a purulent discharge from the preputial opening. Transmissible venereal tumors may affect both the prepuce and the penis. They are single or multiple gray to red masses which often become ulcerated. Biopsy of the Testicles Testicular biopsy is used for studies of histology and karyotype. For taking a tissue sampl~ a small incision is made on the lateral side of the testis after local anesthesia. The tunica albuginea is incised between visible blood vessels. By slight compression of the testis a small piece of tissue will protrude above the surface of the testis. It is cut off with a sharp razor-scalpel and immediately placed in a fixative. For histologic studies Bouin's solution is used (not formalin). For study of the meiotic karyotype, the specimen is treated according to instructions for the examining laboratory. Examination of the Accessory Glands In dogs the normal prostate gland usually can be palpated through the rectal wall. It has two symmetrical lobes and the over-all measurements will normally not exceed 3 to 3.5 em. The hyperplastic gland is usually symmetrical and has a smooth surface. It may be displaced cranially and therefore difficult to reach. In prostatitis the gland is enlarged and the dog experiences pain at palpation. Irregular nodular enlargement is typical of carcinoma, and in this disease the gland often shows adhesion to surrounding tissues and extreme pain is produced by palpation.
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ARTIFICIAL INSEMINATION Although artificial insemination in dogs was first performed about 200 years ago, and an artificial vagina for dogs was developed in the beginning of this century, the method has not become common in breeding work. There are several reasons for this: methods for selection of genetically superior dogs have not been developed; and it is difficult to preserve and transport dog semen. Indications for artificial insemination are: 1. To bridge the distance between the dog and the bitch when they are kept far away from each other; 2. To increase the use of especially valuable dogs; 3. To achieve mating in animals with some traumatic injury which would prevent natural mating; and 4. To prevent the spread of genital infections.
Handling of the Semen The collection and evaluation of the semen have been described previously. If the bitch is present at the time of semen collection, whole semen is used for insemination immediately after collection. Whole semen can be kept for a few hours after cooling on a water bath and kept at 5 to 10° C. For storage beyond a few hours and for transport, the spermrich fraction of the ejaculate should be diluted 1:4 to 1 :8 with an extender. Several extenders have been used. One of the most successful has been heated skim milk. The milk is slowly heated to 92-94° C. (200° F.) and kept at this temperature for ten minutes. After cooling, antibiotics are added before use. Semen diluted in skim milk can be stored for 24 hours witho~t loss of fertility if sufficient sperm are inseminated. One pregnancy has been reported after storage of the semen for five days in this extender. Egg yolk buffered in different ways also has been used as an extender for dog semen, and has given good survival and fertility for at least 24 hours. Several attempts have been made to deepfreeze dog semen. Some authors report a good survival after freezing and storage for long periods. Motility is rapidly lost after thawing, however, and the frozen semen seems to be infertile as a rule. Only one experiment giving pregnancies with deep-frozen semen has been reported. In that experiment the semen was diluted in ll per cent lactose with 4 per cent glycerine and 20 per cent egg yolk and frozen in pellets (Saeger, 1969).
Insemination The correct time to inseminate is the day after the bitch has first accepted the male. The inseminations are repeated once or twice with intervals of 48 hours if the bitch is still showing good "standing" heat. In doubtful cases, especially in bitches with long heat periods, the vagi-
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nal cytology may be examined according to methods mentioned earlier to predict the optimal time for insemination. The bitch is held on a convenient table , and the vulva is cleaned carefully. A 5- or 10-ml. syringe containing the semen is attached to a 9-inch plastic inseminating pipet by a rubber connector. The pipet is inserted into the vagina as illustrated in Figure 4A. As it is advanced to the cervix the pipet is elevated and passed through the cervix if possible (Fig. 4B). The semen is gently injected and the pipet withdrawn. The rear quarters of the bitch are elevated for several minutes. Some authors insert a glove-covered finger into the vagina and "feather"
Figure 4.
Canine artificial insemination technique.
A , T he plastic pipette is inserted anterodorsally to the region of the cervix. B , Seme n is injected slowly and gently.
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Fi gu re 5. Finge r introduced into the vagina to simulate the normal "tie."
or stroke its dorsal wall during the interval the rear quarters are raised (Fig. 5). Conception rates approach those of normal mating if this technique is carefully followed. Preserved semen shipped long distances has not produced such high rates of conception.
REFERENCES l. Cobb, C. M. : Radiographic o utline of genital system of the bitch. Vet. Rec. , 71:66 , 1959. 2. Ford, L.: Canine cytology - an o verlooked clinical technic? Modern Vet. Pr act., 49:3 2, 1968. 3. G)er, H. T.: Estrous cycle in the bitch: Vaginal fluids . Veterinary Scope, 5 , 2 , 1960. 4. Harrop, A. E.: Reproduction in the dog. London, Bailliere, Tindall a nd Cox, 1960. 5. Helper, L. C .: Diagnosis of pregnancy in the bitch with an ultrasonic Doppler instrument. J. A. V. M. A. , 156.60, 1970. 6. Leon ard, E. P. : The Artificial Inse mination of Dogs. ln: The Artificial Insemination of Farm Animals, E . .J. Perry, ed. New Brunswick, Ru tgers University Press, 4th ed., 1968. 7. Saeger, S. W . .J. : Successful pregnancies utilizing frozen dog semen. Artif. Insem. Digest, 17: 12, 1969. 8. Schutte, A. P.: Canine vaginal cytology. J. Small Anim. Pract. , 8 :301, 1967.
Royal Veterinary College 10405 Stockholm Sweden