Canine Reproduction
0195-5616/91 $0.00
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Achieving Canine Pregnancy by Using Frozen or Chilled Extended Semen Catharina Linde-Forsberg, DVM, PhD*
The use of artificial insemination (AI) in dog breeding has experienced a tremendous increase in popularity over the last few years as breeders become increasingly aware of the fascinating possibilities that it offers, and the results of AI are improving as new knowledge and better techniques for semen preparation, estrous detection, and insemination become available. The most suitable stud can now be chosen even if it is thousands of miles away. The age of the stud may not even be a factor if its owner has been foresighted enough to store its genes in a semen bank. Particularly for the less common breeds, access to imported semen can be crucial in avoiding the adverse effects of inbreeding, thereby keeping the breed healthy and sound. In most countries, the kennel clubs, in one way or another, restrict the right to freeze and store dog semen to a few approved agencies with strict record-keeping practices. In this way, the origin of each semen dose can be determined reliably. Actually, the limiting factor today for making full use of the technique is the number of practitioners specializing in small-animal reproduction who are available to help breeders with advice and inseminations. It is hoped that within the next few years many more colleagues will discover and engage in this rewarding field of work. The factors most important in determining the success of artificial insemination in dogs are
l. Timing of the insemination 2. Semen quality and handling 3. Insemination technique THE PROPER DAY FOR INSEMINATION The bitch is unique among the domestic animals with her long period of heat and receptivity. Much knowledge has been gained during the last *Associate Professor, Department of Obstetrics and Gynaecology, Veterinary Medical Faculty, Swedish University of Agricultural Sciences, Uppsala, Sweden Veterinary Clinics of North America: Small Animal Practice-Yo!. 21, No. 3, May 1991
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10 years regarding her reproductive physiology and endocrinology, and excellent review articles on these subjects have been published. 8 • 9 • 49 The long duration of bitches' heat period creates many practical diagnostic problems, especially because the variation between bitches is very pronounced. The canine proestrus, that is, the time from the first day of observed vaginal hemorrhage until readiness to stand for the male dog, averages 9 days but can be as short as 2 to 3 days or as long as 27 days. 60 The estrus, that is, the period of receptivity, qlso averages 9 days but may be as short as 2 to 3 days or as long as 21 days. 13 Thus, some bitches should be inseminated in the first week of their heat, whereas others should not be inseminated until after day 27. Many breeders, however, still stick to the old rule of thumb that bitches should be mated between days 10 and 14. As a result, bitches with divergent but normal heat periods will not conceive. The breeders often need help to diagnose those extreme heat periods. It is clear that it is not possible to determine the fertile days in the heat period accurately if timing is based on the first day of proestrus. On the other hand, the bitch can become pregnant after a single mating from about 1 day before until about 7 or 8 days after luteinizing hormone (LH) peak. 35 • 60 These assumptions are based on findings that the semen maintains its longevity for about 4 to 7 days, 15• 18 • 58 the LH peak precedes ovulation by 36 to 50 hours, 11 • 50 and the ova undergo maturation divisions for another 2 to 5 days before obtaining the capacity to become fertilized 4 • 31 · 57 and remain alive for 1 to 4 days thereafter. 13· 58 The capacitation time of canine spermatozoa has been estimated to be 7 hours. 41 Thus, theoretically the optimum time for mating would be around day 4 after the LH peak when the ova are shed and most of them have completed maturation division (Fig. 1). Diagnostic Methods Clinical Signs. To determine the optimum days for inseminating the bitch, the clinical signs of vulvar swelling and vaginal discharge and the Optimal mating time
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DAYS FROM LH-PEAK Figure 1. Temporal relationship between LH peak, ovulation, maturation of ova, and the hypothetically maximal timespan for successful mating of the bitch. (From Linde C, Karlsson 1: The correlation between the cytology of the vaginal smear and the time of ovulation in the bitch. J Small Anim Pract 25:77, 1984; with permission.)
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behavior of the bitch when teased with a male dog can be used. Usually, the swelling of the vulva has receded somewhat, hemorrhaging has diminished, the discharge is more straw-colored, and, ideally, the bitch has already allowed the male dog to mount her for a couple of days. The owner should be asked to supply these details. During insemination, palpation to determine the size and consistency of the cervix also provides clues as to the stage of estrus, because the cervix responds to changing levels of estradiol in a manner similar to that of the vulva. That is, during proestrus and early estrus it grows large and hard, and during late estrus it diminishes in size and becomes softer. Vaginal Examinations. A quick and simple diagnostic method to determine the best time for insemination is to take a vaginal smear and determine the degree of cellular cornification under a microscope. 7 • 35· 52 This will give the experienced examiner a good indication as to whether or not the bitch is in proper estrus. Because the cellular changes are caused by changes in estradiol, however, no firm conclusions as to whether ovulation has taken place can be made. In the dog, there have not been any reports of specific cellular changes in the vaginal smear being caused by the rise in progesterone following ovulation. For mating or insemination with fresh semen, which is expected to retain its fertilizing capacity in the uterus of the bitch for between 2 and 11 days, 15· 18· 58 the vaginal smear still is very useful. However, when using frozen semen, which has a comparatively short postthaw survival time, more specific methods are required to determine when the ova are shed and ready to be fertilized in the oviduct. 36 The endoscopic appearance of the vaginal vault during different stages of the estrus cycle has been described. 37 • 38 This method may prove very useful for clinicians who have access to an endoscope. Strips for measuring the vaginal glucose content63 or the electrical resistance of the vaginal mucus 29 have been evaluated, but both methods suffer from the same limitations as the vaginal smear: they reflect the effects of estradiol but not of progesterone. Changes in LH and Progesterone During the Estrous Cycle. Changes in LH levels during the estrous cycle of the dog have been reported by several researchers. 5 · 6 • 11 • 12• 45 • 47 • 56 LH levels that are low during proestrus increase 20- to 40-fold during the preovulatory surge at the end of proestrus. The preovulatory rise in LH lasts about 24 to 72 hours and causes ovulations 36 to 50 hours after the LH surge. LH release patterns in the dog, as in other mammalian species, are pulsatile. LH concentrations are 0.2 to 1.2 ng/mL between pulses during anestrus, reach levels of 2 to 25 ng/mL, average 5 ± 1 ng/mL during episodic pulses, and peak at 8 to 50 ng/mL with an average of 20 ng/mL during the preovulatory LH surge. 13· 14 Progesterone levels in the bitch are less than 0.5 ng/mL (25 ng/mL (>78,5 nmol/L). (Fig. 2). Hormone Assays. The most accurate methods available for determining the time of ovulation in the bitch involve measuring peripheral plasma
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Figure 2. Schematic representation of the hormonal events during the heat period in the bitch and the ideal time for insemination.
levels of LH or progesterone. Recently a rapid radioimmunoassay (RIA) for LH analysis has been developed that will give a result within 3 hours. The results of this method are comparable to those of the much longer standard LH assay. 40 The advantage with the LH assay is that the breeder will know several days in advance when to inseminate the bitch, because the LH peak precedes ovulation (see earlier discussion). The disadvantage is that the LH peak in the dog has a duration of only 1 to 2 days, which necessitates daily blood sampling (for the rapid RIA) or sampling every other day (for the long RIA) and frequent analyses. There are several RIA and ELISA methods available for progesterone analysis. 17• 19• 21 • 32 Most of the commercially available ELISA kits are semiquantitative as they are adapted for use in other species. Thus, they are too unspecific to be of help. Using such kits, one can determine whether the peripheral plasma progesterone is below or above a certain level, usually around 7 ng/mL (22 nmoi/L). Because the shift in color indicating the higher level of progesterone is difficult to discern with the naked eye, a photometer may be needed, and such instruments are expensive. 17 The more quantitative ELISA kits require micropipetting of very minute volumes (20 !J.L) and demand a great deal of routine laboratory practice to give accurate and repeatable results. Thus, they are not as practical as they may seem at first. With a sensitive RIA technique, the progesterone level can be given with an inter- and intra-assay variation of not more than 10% at plasma levels above 3 ng/mL (10 nmol!L). 22 • 32· 39 The assay takes from 6 to 24 hours. With the Coat-A-Count (Diagnostic Products Corp., Los Angeles, California) method the analysis may take no longer than 3 hours. 22 Therefore, the practitioner is advised to establish contact with a good
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referral laboratory for canine progesterone analysis. If the sample is posted immediately after collection, the results should be available over the telephone the following afternoon. When to Inseminate Studies have shown that inseminations with fresh semen performed so early that the progesterone level was still below 5,44 ng/mL (17,1 nmol/L); that is, before ovulation, often may result in pr~gnancy. With frozen-thawed semen, however, which has a shorter postthaw survival time, only one pregnancy resulted from 15 inseminations performed at a progesterone level below 9.6 ng/mL (30 nmol!L). 36 Ideally, the progesterone level should range between 10 and 25 ng/mL (30 and 75 nmol/L), which means that the bitch should be inseminated late in estrus (Fig. 2). The most practical way to effect this is to estimate the correct day with the aid of the details given by the owner of the bitch. Then, the bitch should be brought in for sampling 1 or 2 days before this estimated date. If it is still slightly early, the progesterone probably has gone up from baseline levels, and the proper day can be calculated from the value obtained by the RIA progesterone assay. On the other hand, if the bitch is late and her progesterone already at >25 ng/mL (>78.5 nmol/L), a vaginal smear must be taken to find out whether she is already in metestrus. Because the maximum level of progesterone may be maintained for several weeks, progesterone levels in these cases cannot be used to estimate the time elapsed since ovulation.
SEMEN QUALITY AND HANDLING Semen Collection Canine semen is easily collected by manual massage. Most dogs are very cooperative. If they are timid or feel uncomfortable in the surroundings, it may help if the veterinarian attempting collection is not wearing a white coat, which may bring about unpleasant memories. The collection should take place with the dog standing on the floor. A bitch in heat may be helpful but usually is not necessary. Her presence, however, might result in a higher total number of spermatozoa in the ejaculate. Dogs' ejaculate is distinctly fractionated . The first fraction is watery, emanates from the prostate gland, and usually has a volume of 1 to 5 mL. It takes around 0.5 to 1 minute to complete. The second fraction is milkish white, 1 to 3 mL in volume, and contains the major portion of the spermatozoa. It takes around 1 to 2 minutes to complete. The third fraction comes from the prostate, constitutes the major portion of the ejaculate, and may be up to 30 to 40 mL in the larger breeds. It may take from around 5 minutes to 1 hour to complete. For insemination purposes, only the second, sperm-rich fraction is of interest. A gloved hand is introduced from behind between the hindlegs of the dog and the caudal part of the bulbus glandis is stimulated by manual massage. When semi-erection is achieved, the preputial sheath is slid backwards so that the bulbus glandis is freed and the penis may be turned
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around allowing collection to take place behind the dog. With this technique, the glans penis and the preputial lining may be inspected for abrasions, wounds, or signs of infection. Also, if a bacterial sample is to be collected, contamination by dust or hair from the dog's coat is avoided (Fig. 3). For semen collection, some people use a rubber cone with a tube attached to the end whereas others slip a plastic bag over the dog's penis. With these methods, however, pus from the prepuce may contaminate the semen sample. Moreover, fractioning of the ejaculate is difficult or impossible. Semen also can be collected in a pre-warmed, calibrated glass or plastic vial equipped with a wide opening and a tapering end. By holding the vial in the closed hand, the semen is protected from changes in temperature. During the pelvic thrusting, the vial should be held at a distance from the penis to avoid trauma and possible hemorrhage. If the dog is reluctant to ejaculate, the thumb of the hand holding the vial can
Figure 3. Semen collection technique in the dog. The penis is flexed backwards. and collection takes place behind the dog.
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be introduced into the fossa of the glans and pulsatile pressure can be applied to the urethral process against the wall of the vial (Fig. 4): Most dogs like this, although some may react adversely. If the first, watery semen fraction is large or fouled by urine or blood, the collecting vial should be emptied by rapidly turning it upside-down and the collection continued without interruption and without distracting the dog. If the semen is very dilute or if the dog has prostatitis, the sample should be gently centrifuged for 5 minutes at 300 G to 700 G24 and the supernatant seminal plasma discarded. Then, the semen is diluted to the desired density and volume with an extender and, in the case of infection, with antibiotics. In this way, both semen quality and longevity can be substantially enhanced. Semen Quality No comprehensive minimum requirements for dog semen to be fertile are available, largely because the reproductive pattern of this species and the way dogs are kept and bred makes them unsuitable for systematic studies. Most published studies therefore are based on clinical material and on inseminations of patients. It should be kept in mind that fertility is not an ali-or-none trait. Poor semen quality influences mean pregnancy rates and mean numbers of puppies based on a large number of matings. But on the individual level, poor quality semen may still result in pregnancy and normal litter size. Most researchers agree that the total number of spermatozoa per insemination should be between 150 to 200 X 106 • 2 • 3• 43 On the other hand, good pregnancy results have been achieved using as few as 20 X 106 fresh spermatozoa deposited surgically at the tip of the uterine horn. 61 This is in agreement with findings by Fougner and Forsberg27 who achieved normal pregnancy and litter size in vixens inseminated into the uterus with as few
Figure 4. Applying pressure on the processus urethralis against the wall of the collecting vial may stimulate reluctant dogs to ejaculate.
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as 20 X 106 fresh spermatozoa. No comparable studies have been made with frozen dog semen. The proportion of spermatozoa with primary and secondary defects should not exceed 30% to 40%. 20· 25 • 33 On the other hand, some found that the proportion of spermatozoa judged to be normal ranged between 0% to 96% among 167 normal dogs. However, the criterion for normality in this study was that the dogs had sired a litter during the last 12 months. 44 A German Shepherd that was used for approxi!,llately 100 matings per year had 60% defective spermatozoa (excluding distal protoplasmic droplets) and a total sperm count of 1.300 x 106 . About half of the bitches mated to this dog did conceive (Linde-Forsberg, unpublished data). Again, the relative significance of different types of defects has been studied very little. It generally is agreed that distal protoplasmic droplets lack significance, although they may be a signal of an ongoing disturbance. Motility is said to be an important parameter in judging canine semen quality and should exceed 70% in a normal semen sample. When dealing with insemination in dogs, semen that has been extended and possibly frozen usually is used. Because some semen extenders reduce motility, it is difficult or impossible to make a judgement unless the composition of the extender is known . Pregnancy was obtained and eight puppies were eventually produced with imported fresh extended semen that had a motility of only 10%. 36 The composition of the extender in this case was unknown. With frozen semen, pregnancies were obtained with semen with a postthaw motility between 20% to 30%. 36 When judging sperm motility, the semen sample should always be allowed to reach room-temperature (37°C is preferred). A good indication of its quality is also obtained by looking at its resistance, that is, the length of time that it retains motility. Morton and Bruce44 compared the longevities of fresh semen stored at 4°C without extender with a Tris-extender and frozen-thawed semen with an extender. The results clearly show that the frozen-thawed fresh semen has the shortest survival time and that the best results, under these conditions, were achieved for fresh-extended semen. 44 Handling of Fresh Semen Semen handling is crucial to achieve good results. Canine semen may survive cooling to 4°C for several days. 44 It is, however, important that the cooling process is gradual (30 to 60 minutes). The most important step in the cooling procedure is that from body temperature to room temperature. This may increase the longevity of the spermatozoa sixfold. Unless the semen can be cooled under well-controlled conditions, it is better to store it at room temperature. In other words, it is better not to chill the semen than to chill it too fast or too much. If the semen is stored with ice, it is imperative that the semen vial is well-insulated so that there is no direct contact with the ice cubes; otherwise, the temperature can drop so low that the spermatozoal membranes disintegrate, leading to enzyme leakage. Although such spermatozoa may show good progressive motility when examined under a microscope after warming them up, they have no fertilizing capacity. At least 15 minutes should be allowed for the subsequent warming of the semen to room temperature before use. Always try to avoid temperature fluctuations.
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When semen is to be·chilled and shipped it should always be extended. The extender helps to keep the spermatozoal membranes from being harmed by changes in temperature and shaking during transport while also providing energy and stabilizing the pH. Morton and Bruce44 clearly showed that the survival time of spermatozoa in extended semen is considerably higher than that of undiluted semen stored at 4°C. Progressive motility after 24 hours was 91.7% for undiluted semen and 98% for diluted semen. After 48 hours, the motility of the undiluted semen was only 36.7% whereas that of the diluted semen was 90%. After 72 hours, the motility of the undiluted semen was down to 18.3%, whereas after 96 hours, the diluted semen still showed 80% motility. 44 Seminal Extenders There are many types of extenders for canine semen. Some of the first described consisted of pasteurized milk: 30 The Illinois Variable Temperature (1. V. T.) extender, 61 and 1.16% sodium citrate dihydrate, 0. 75% glycine, and 1% glucose with 20% egg yolk (v/v). 26 Today, one of the most commonly used extenders is a Tris-citrate buffer with 20% egg yolk and antibiotics. 16 The author's favorite extender for fresh semen consists of 80% homogenized and pasteurized cream (12% fat) and 20% (v/v) egg yolk. One mg of dihydrostreptomycin and 1000 IU of benzyl penicillin are added per mL of final solution. For an average ejaculate consisting of 1 to 2 mL of spermdense, second-fraction semen, 4 mL of pasteurized cream is mixed with 1 mL of egg yolk. The egg yolk should be carefully separated from the white by placing the former on a filter paper and gently rolling it around until the white has come off. Use a 2-cc disposable syringe to measure the correct volume of egg yolk. Before mixing the extender and the semen, make sure that the two solutions are at the same temperature. The dilution rate usually ranges from 1:3 to 1:5. The final volume of extended semen should not exceed 5 to 10 mL per insemination dose. Chilling of Fresh Semen
If the semen is to be chilled, place the semen vial (preferably a sterile plastic tube with a screw cap that will not break during transport [e.g., a Nunc-tube]) in a glass filled with room-temperature water before putting it in the refrigerator. Leave the tube in the refrigerator for 30 to 60 minutes until the temperature has dropped to the desired level, remembering not to chill it too fast or to too low a level. For most transport distances, a temperature between 10° to 15°C should be sufficient. At the same time, put the thermos bottle filled with moistened cotton wool as shock absorbant in the refrigerator so that it will be the same temperature as the semen sample. Semen prepared in this manner will maintain its fertilizing capacity for at least 12 to 24 hours and usually longer. Frozen Semen
If dog semen is to be frozen, the extender must also contain glycerol. It is not within the scope of this article to describe freezing techniques,
because most practitioners are not in a position to freeze semen. For
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those interested, several papers and review articles have been published. 2. 10, 44, 53, 54, ss Thawing of Frozen Semen. Frozen semen should always be thawed as recommended by the person freezing it, because the method of choice depends on the method of freezing. Rapid freezing requires rapid thawing and vice versa. This information has to be supplied by the freezing agency. The semen can be frozen either in pellets or in straws (usually 0.5 mL but occasionally 0.25 mL). The pelleted semen is always thawed in a thawing medium, usually saline or citrate, at 37°C. Semen frozen in straws may be thawed with or without thawing media. The 0.5-mL straws are thawed in a 37°C water bath for 15 seconds and a thawing medium is used (Govette, personal communication), or bathed for 60 seconds, 43 or in a water bath of 75°C for 6.5 seconds, 1. 2 or for 12 seconds. 46 For mini-straws (0.25 mL), the thawing time should be reduced by approximately one half. Fresh or Frozen Semen? Most canine inseminations today are performed with fresh semen. It is easy to handle and ship, and the insemination technique used is relatively simple. Fresh, usually extended and chilled, canine semen may, if of originally good quality, retain its fertilizing capacity for at least 12 to 24 hours, usually considerably longer. This means that it can be used for most national shipments, and for many international ones as well. The author, who practices in Europe, considers the advantages of using fresh semen to outweigh the disadvantages in almost every case, especially because results thus far are better than for frozen semen. A study of the results of 470 inseminations showed that both pregnancy rate and litter size are higher for bitches receiving fresh semen. 36 The pregnancy rate was 83.8% with good quality fresh, chilled extended semen compared with 69.3% with good quality frozen semen. Litter size was estimated to be 23.3% smaller in the group of bitches receiving frozen semen, or 29.7% on a within-breed basis. 36 In a smaller number of inseminations, Farstad 23 obtained a pregnancy rate of 84% with fresh and 67% with frozen semen but no reduction in litter size with frozen semen. 23 Fresh semen may be shipped in an ordinary thermos bottle, preferably one with a wide opening. It may be forwarded by air as an ordinary parcel at a modest rate. The transport thermos is of no great value and need not be returned to the sender. (It obviously can be kept by the buyer to be used for its intended purpose.) The insemination can be made in the cranial vagina, which is technically quite easy for a trained inseminator. The disadvantage with fresh semen is that everything has to be arranged on the day most suitable for the bitch. Usually at least four people have to be available at a certain date. The owner of the stud dog has to contact his or her veterinarian for the semen collection and book a convenient flight. At the same time, the owner of the bitch has to arrange with his or her veterinarian to have the insemination done. Thus, the potential for encountering problems is high, especially if the date in question is a holiday. The advantages with frozen semen are that it can be shipped at a time convenient for all parties and it can be available when it is needed. Moreover, only two people have to be available on the chosen day, the
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bitch owner and the inseminating veterinarian. Also, if the semen is shipped a long distance or even imported, many doses can be sent in a single shipment to be used when desired. The semen banking option certainly will prove to be of exceptional value in dog breeding because the semen may be stored practically forever. The disadvantages of frozen semen have already been mentioned: Only a limited number of technicians have mastered the freezing technique, and the kennel clubs only allow it to be performed .by a few people and agencies. Frozen semen usually has to be shipped as dangerous goods with all the extra charges that this entails. This, however, may be avoided by using one of the modern dry-shippers. These liquid nitrogen containers absorb the nitrogen into their walls, thereby preventing spillage even if the container is turned on its side or upside-down. This also means that the hazards of ruining the semen during transport are minimized. For those involved in this business, an investment in this modern equipment is highly recommended. The container is expensive and usually of no further use to the buyer of the semen and is thus returned to the shipper, which entails some extra costs. The insemination technique using frozen semen is considerably more difficult than that used for fresh semen inseminations (see later discussion). Depositing frozen semen in the cranial vagina generally results in a very poor pregnancy rate. 2• 3 • 36 In conclusion, the use of fresh-extended and chilled semen is usually the method of choice because it is easy, less expensive, and gives a better result. With good planning, commencing as soon as the bitch comes into heat, the problem of organizing the collection and shipment of semen and the insemination should not be too difficult to overcome. In most cases, the longevity of the semen is good enough to allow for transport times of at least 12 to 24 hours. Frozen semen should be recommended when the transport times are too long, when semen for more than one bitch is requested, when the people involved are not available on the best day for insemination of the particular bitch, and, obviously, when semen is to be banked for future use. INSEMINATION TECHNIQUES The current methods for inseminating bitches include vaginal deposition, transcervical intrauterine deposition, surgical intrauterine deposition, and intrauterine insemination by laparoscopy. In many countries, the latter two methods are considered to be unethical and they are not discussed further in this article. Insemination Catheters For vaginal inseminations, a 45 x 0.5 em disposable plastic catheter is used (originally for uterine infusions in the bovine). For transcervical intrauterine inseminations, a 20- to 50-em-long steel catheter with a 0.5mm to 1-mm diameter tip is used together with a protecting nylon sheath (Fig. 5). The latter is a Norwegian construction adapted from a model used
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Figure 5. Insemination catheters. Top, the plastic catheter used for deep vaginal inseminations in the bitch. Below, three sizes of the Norwegian catheter for intrauterine inseminations. A 5- to 10-cc syringe should be used for the semen.
for inseminations of farmed foxes. 2 Usually, three different lengths of the catheter are sufficient for insemination of most breeds. There also is a modified version of the latter catheter that has an outer metal sheath with a loop at the end intended to fit around, and thus fixate, the portio vaginalis of the cervix. 28 The advantage of this model is that once the cervix is fixed in the loop, it may be rotated easily to facilitate palpation and introduction of the inner catheter through the cervical canal. The disadvantage is that roughly 10 different sizes of loops are necessary to cover the variation in size of the portio vaginalis among dog breeds. A French company is marketing a flexible vaginal catheter with an inflatable cuff intended to be left in situ for some time after insemination. 42 Thus far, there are no published data indicating that results with this equipment are superior to those obtained with the standard deep-vaginal insemination of fresh semen or the transcervical insemination of frozen semen followed by holding the hindquarters of the bitch elevated for 10 minutes. Abdominal Palpation The clinician conducting canine inseminations should learn to locate the cervix of the bitch through abdominal palpation in order to be able to deposit the semen in the correct place . Correct placement is as crucial for vaginal inse minations as for intrauterine inseminations. From the size and consistency of the cervix the clinician can also tell whether the bitch is at the right stage of the estrous cycle (see earlier discussion). Because the uretheral opening of the bitch is located at the brim of the pelvic floor, it is surprisingly easy for the catheter to be unintentionally introduced into the urinary bladder. If the bladder is void of urine or if the syringe containing the semen is applied to the end of the catheter, no urine is seen in the catheter to indicate its position in the bladder. Apart from the hazard of perforating the bladder with the catheter, it is obvious that no pregnancy
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will follow. Thus, the correct position of the tip of the catheter should always be checked by palpation before depositing the semen dose. Vaginal Insemination The bitch to be palpated or inseminated should have an empty stomach to facilitate the procedure. To locate the cervix, the plastic catheter is introduced into the vagina of the bitch. No speculum is needed. The angle of introduction is either steeply upward until the pelvic brim has been passed, or the vulva is elevated until it is just below the anus (as the bitch does when inviting the male dog) and the catheter is introduced straight ahead. The vagina then slopes slightly forward and downward (Fig. 6). When the tip of the catheter is in front of the pelvic floor, it is palpated to help orientation. By elevating the distal end of the catheter, the tip is lowered closer to the abdominal wall and thus becomes more accessible to palpation. The catheter is carefully introduced until it reaches the narrow, cranial portion of the vagina created by the dorsal, median postcervical fold 51 (see Fig. 6). In some bitches, especially maiden ones or ones of small breeds, it is not possible to pass the catheter all the way through to the cervical fornix. The cranial vagina can be felt as a 1- to 2-cm-long, firm structure that ends at the cervix, which in a bitch in estrus is a 0.5- to 1.5cm hard, rounded to ovoid structure that is freely movable. The corpus uteri and the uterine horns can be felt in front of the cervix. To palpate them, work forward with the aid of the catheter by lowering its tip, closing the tip of the thumb against that of the index finger above the catheter; and then lifting its tip so that the cervix or the uterine horns are forced
Figure 6. Colpogram of an estrous bitch clearly outlines the narrow cranial part of the vagina and the fornix surrounding the estrogen stimulated cervix (on the left). (Courtesy of Anne-Sofie Lagerstedt, DVM.)
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upward between the fingers (Fig. 7). Their size and consistency then become evident. This method of palpating the uterus also is very useful in examining a bitch with suspected endometritis or pyometra and for early pregnancy detection from 3 to 5 weeks after mating. Because the plastic catheter is much too wide to be able to pass through the normal cervical canal, there is no risk involved for the fetuses. If the catheter is in the urinary bladder, the thick, cranial part of the vagina and the cervix are palpable above the catheter. In addition, because the walls of the urinary bladder usually are much thinner than those of the vagina, the tip of the catheter stands out more clearly. Even though the urine usually is sterile, it is recommended that the catheter be exchanged for a new one if the bladder has been mistakenly catheterized. Intrauterine Insemination In transcervical intrauterine insemination, the steel catheter is introduced into the vagina, which is protected by the nylon sheath. Once the pelvic floor is passed, the cranial end of the nylon sheath is palpated as described earlier. It usually is too wide to be introduced into the cranial, narrow part of the vagina. Before its introduction, a mark is made on the distal part of the steel catheter to indicate when its tip is in front of the nylon sheath. The sheath is palpated, and the cervix usually is found 1 to 2 em in front and slightly above the sheath if the tip of the catheter has been lowered closer to the abdominal wall. The steel catheter then is introduced through the nylon sheath all the way into the fornix, that is, below the portio vaginalis of the cervix. The cervix is fixed through the
Figure 7. Abdominal palpation in a bitch with the aid of a vaginal catheter. By lowering the tip of the catheter (on the left}, the cranial vagina, cervix, and caudal part of the uterine horns become accessible for palpation.
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abdominal walls between the thumb and the index finger and, by a slightly downward traction at the corpus uteri, it is tilted so that the angle of the cervical canal is changed from between 45° to 60° to a more horizontal angle (Fig. 8). The tip of the catheter is then moved, using very small movements caudally and touching against the cervix in search of the opening of the cervical canal. The sensation when the opening is found can best be described as the sensation of touching cartilage, that is, "crispy." Once the opening has been found, fix the catheter anel start working the cervix against the catheter. The cervical canal may be some 5 to 10 mm long, and is not always straight. Thus, it often has to be slightly forced onto the catheter. In most bitches, the tip of the catheter easily can be felt in front of the cervix in the corpus uteri. In some bitches, however, the sensation is not as distinct. For training purposes, saline can be infused to check the correct position of the catheter in the uterus of the bitch. If the catheter is in the right position, the fluid can easily be infused, and if air is present in the catheter or syringe, as is often the case, a characteristic, bubbling sound may be heard when introducing the fluid through the cervix. If, on the other hand, the catheter still is in the vagina, there will be a backflow between the catheter and the nylon sheath. Another way to control the position of the tip of the catheter during training is to infuse 60% U rographin or Amipaque and to radiograph the bitch. The bitch should be held with elevated hindquarters for at least 10
A
Figure 8. Sagittal section of the canine cranial vagina and cervix with the Norwegian intrauterine insemination catheter in position. A, The cervical canal is at an angle of approximately 45° to 60° to the cranial vagina. B, To facilitate catheterization, the cervix is fixed between the thumb and index finger by abdominal palpation and tilted in a more horizontal position.
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minutes after insemination with fresh or frozen semen to help the uterine transport of spermatozoa toward the oviducts34 (Fig. 9). Studying bitches with experimental uterine fistulas, Tsutsui59 found semen at the tip of the uterine horn about 30 seconds to 1 minute after a natural mating and about 30 seconds to 2 minutes after vaginal insemination when the bitch was held with elevated hindquarters. When the bitch was inseminated standing horizontally, the transport of spermatozoa was greatly hindered. When fresh semen is inseminated, the hindquarters of the bitch are elevated with the catheter in position in the cranial vagina. The bitch should be held so that the catheter is in a vertical position before infusing the semen (Fig. 9). After deposition of the semen, the catheter is withdrawn, and the vulva is feathered to induce the bitch to flag her tail and to stimulate uterine contractions, which supposedly facilitate sperm transport. The frozen-thawed semen is introduced with the bitch standing in the normal position, and the cervix fixed between the fingers. After deposition of the semen, the hindquarters are elevated. The catheter is then withdrawn, and the bitch is held as described earlier. Sedation is usually not
Figure 9. It is important to hold the bitch with its hindquarters elevated after deposition of the semen. The catheter (left in to demonstrate the desired angle) is removed and the perivulvar region feathered to improve uterine transport of spermatozoa.
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needed; on the contrary, most bitches in estrus like this type of handling. Remember to instruct the owner that the bitch should have an empty stomach to facilitate palpation. Insemination Volume Because the uterine lumen in the bitch is quite small, insemination volume should not exceed 5 to 10 mL. Diluting the semen more results in a back-flow of spermatozoa as soon as the ~itch is back on all four feet again. How Many Times Should the Bitch Be Inseminated? Repeated inseminations with either fresh or frozen semen have been reported to result in higher pregnancy rates than single inseminations. 24 • 36 This is probably a result of the extended period of ovulation and maturation of the ova in the bitch. If for practical or economic reasons only one insemination can be made, emphasis should be placed on making sure that the bitch is inseminated at the optimum time, rather late in estrus.
REFERENCES 1. Aamdal J, Andersen K: Fast thawing of semen frozen in straws. Zuchthyg 3:22, 1968 2. Andersen K: Insemination with frozen dog semen based on a new insemination technique. Zuchthyg 10:1, 1975 3. Andersen K: Artificial insemination and storage of canine semen. In Morrow DA (ed): Current Therapy in Theriogenology: Diagnosis, Treatment and Prevention of Reproductive Diseases in Animals. Philadelphia, WB Saunders, 1980, p 661 4. Andersen AC, Simpson ME: The Ovary and Reproductive Cycle of the Dog (Beagle). Los Altos, Geron-X Press, 1973 5. Chakraborty PK: Reproductive hormone concentrations during estrus, pregnancy, and pseudopregnancy in the Labrador bitch. Theriogenology 27:827, 1987 6. Chakraborty PK, Panko WB, Fletcher WS: Serum hormone concentrations and their relationships to sexual behavior at the first and second estrous cycles of the Labrador bitch. Bioi Reprod 22:227, 1980 7. Christie DW, Bailey JB, Bell ET: Classification of cell types in vaginal smears during the canine estrus cycle. Br Vet J 128:301, 1972 8. Concannon PW: Clinical and endocrine correlates of canine ovarian cycles and pregnancy. In Kirk RW (ed): Current Veterinary Therapy IX. Philadelphia, WB Saunders, 1986, p 1214 9. Concannon PW: Canine physiology of reproduction. In Burke TJ (ed): Small Animal Reproduction and Infertility. A Clinical Approach to Diagnosis and Treatment. Philadelphia, Lea & Febiger, 1986, p 23 10. Concannon PW, Battista M: Canine semen freezing and artificial insemination. In Kirk RW: Current Veterinary Therapy X: Small Animal Practice. Philadelphia, WB Saunders, 1989, p 1247 11. Concannon PW, Hansel W, McEntee K: Changes in LH, progesterone and sexual behavior associated with preovulatory luteinization in the bitch. Bioi Reprod 17:604, 1977 12. Concannon PW, Hansel W, Visek WJ: The ovarian cycle of the bitch: Plasma estrogen, LH and progesterone. Bioi Reprod 13:112, 1975 13. Concannon PW, McCann JP, Temple M: Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J Reprod Fertil [Suppl] 39:3, 1989 14. Concannon PW, Whaley S, Anderson SP: Increased LH pulse frequency associated with termination of anestrus during the ovarian cycle of the dog [abstract]. Bioi Reprod 34 (Suppl):ll9, 1986
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15. Concannon PW, Whaley S, Lein D et al: Canine gestation length: Variation related to time of mating and fertile life of sperm. Am J Vet Res 44:1819, 1983 16. Davis JS, Bratton RW, Foote RH: Livability of bovine spermatozoa at 5°, -25° and -85° Cin Tris-buffered and citrate-buffered yolk-glycerol extenders. J Dairy Sci 46:57, 1963 17. Dee J, Forchhammer S: Progesteronmaling hos hund. Fastsaettelse af parringstidspunkt. Dansk VetTidsskr 71:425, 1988 18. Doak RL, Hall A, Dale HE: Longevity of spermatozoa in the reproductive tract of the bitch. J Reprod Fertil 13:51, 1967 19. Eckersall PD, Harvey MJA: The use of a bovine plasma progesterone ELISA kit to measure progesterone in equine, ovine and canine-plasmas. Vet Rec 3:5, 1987 20. England GCW, Allen WE: Seminal characteristics and fertility in dogs. Vet Rec 124:399, 1989 21. England GCW, Allen WE, Porter DJ: A comparison of radioimmunoassay with quantitative and qualitative enzyme-linked immunoassay for plasma progestogen detection in bitches. Vet Rec 124:107, 1989 22. Epelu-Opio J, Madej A: Direct measurement of progesterone in plasma and milk by a simple solid-phase radioimmunoassay. Vet Clin Pathol17:26, 1988 23. Farstad W: Bitch fertility after natural mating and after artificial insemination with fresh or frozen semen. J Small Anim Pract 25:561, 1984 24. Farstad W, Andersen-Berg K: Factors influencing the success rate of artificial insemination with frozen semen in the dog. J Reprod Fertil [Suppl] 39:289, 1989 25. Feldman EC, Nelson RW: Disorders of the canine male reproductive tract. In Canine and Feline Endocrinology and Reproduction. Philadelphia, WB Saunders, 1987, p 481 26. Foote RH, Leonard EP: The influence of pH, osmotic pressure, glycine and glycerol on the survival of dog sperm in buffered yolk extenders. Cornell Vet 54:78, 1964 27. Fougner JA, Forsberg M: Effect of different sperm numbers on fertility after artificial insemination of foxes . Acta Vet Scand 28:403, 1987 28. Funkquist B, Lagerstedt A-S, Linde C, et al: Hysterography in the bitch. Vet Radio! 26:12, 1985 29. Giinzel A, Koivisto P, Fougner J: Electrical resistance of vaginal secretion in the bitch. Theriogenology 25:559, 1986 30. Harrop AE: Artificial insemination of a bitch with preserved semen. Br Vet J 110:424, 1954 31. Holst PA, Phemister RD: The prenatal development of the dog: Preimplantation events. Bioi Reprod 5:194, 1971 32. Kindahl H, Edqvist L-E, Granstrom E et al: The release of prostaglandin F 2a as reflected by 15-keto-13,14-dihydroprostaglandin F 2a in the peripheral circulation during normal luteolysis in heifers. Prostaglandins 11:871, 1976 33. Larsen RE: Evaluation of fertility problems in the male dog. Vet Clin North Am 7:735, 1979 34. Linde C: Transport of radiopaque fluid into the uterus after vaginal deposition in the oestrous bitch. Acta Vet Scand 19:463, 1978 35. Linde C, Karlsson I: The correlation between the cytology of the vaginal smear and the time of ovulation in the bitch. J Small Anim Pract 25:77, 1984 36. Linde-Forsberg C, Forsberg M: Fertility in dogs in relation to semen quality and the time and site of insemination with fresh and frozen semen. J Reprod Fertil [Suppl] 39:299, 1989 37. Lindsay FEF: The normal endoscopic appearance of the caudal reproductive tract of the cyclic and non-cyclic bitch: Post-uterine endoscopy. J Small Anim Pract 24:1, 1983 38. Lindsay FEF, Concannon PW: Normal canine vaginoscopy. In Burke TJ (ed): Small Animal Reproduction and Infertility. A Clinical Approach to Diagnosis and Treatment. Philadelphia, Lea & Febiger, 1986, p 112 39. Madej A, Kindahl H, Nydal C, et al: Hormonal changes associated with induced late abortions in the mare. J Reprod Fertil [Suppl] 35:479, 1987 40. Madej A, Linde-Forsberg C, Garnum F: A rapid radioimmunoassay for determination of LH in dogs [abstract]. J Reprod Fertil [Suppl] 39:329, 1989 41. Mahi CA, Yanagamachi R: Maturation and sperm penetration of canine oocytes in vitro. J Exp Zool196:189, 1976 42. Mialot J-P, Dumon C, Cassou B: Artificial insemination in bitches: Introduction of fresh
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semen with the Osiris flexible gun. Practique Medicale et Chirurgicale de l'Animal de Compagnie 20:213, 1985 Morton DB: Artificial insemination with frozen semen in the dog: Principles of 'DNA fingerprinting.' In Jones DE, Joshua JO (eds): Reproductive Clinical Problems in the Dog. London, Wright, 1988, p 169 Morton DB, Bruce SG: Semen evaluation, cryopreservation and factors relevant to the use of frozen semen in dogs. J Reprod Fertil [Suppl] 39:311, 1989 Nett TM, Akbar AM, Phemister RD, et al: Levels of luteinizing hormone, estradiol and progesterone in serum during the estrous cycle and pregnancy in the Beagle bitch. Proc Soc Exp Bioi Med 148:134, 1975 Olar TT: Cryopreservation of dog spermatozoa [dissertation]. Fort Collins, CO, Colorado State University, 1984 Olson PN, Bowen RA, Behrendt M, et al: Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus and estrus. Bioi Reprod 27:1196, 1982 Olson PN, Bowen RA, Behrendt M, et al: Concentrations of progesterone and luteinizing hormone in the serum of diestrous bitches before and after hysterectomy. Am J Vet Res 45:149, 1984 Olson PN, Nett TM: Reproductive endocrinology and physiology of the bitch. In Morrow DA: Current Therapy in Theriogenology 2. Philadelphia, WB Saunders, 1986, p 453 Phemister RD, Holst PA, Spano JS, et al: Time of ovulation in the Beagle bitch. Bioi Reprod 8:74, 1973 Pineda MH, Kainer RA, Faulkner LC: Dorsal median postcervical fold in the canine vagina. ,Am J Vet Res 34:1487, 1973 Roszel JF: Genital cytology of the bitch. Scope XIX 1:1, 1975 Seager SWJ: Successful pregnancies utilizing frozen dog semen. AI Digest 17:26, 1969 Seager SWJ, Fletcher WS: Progress on the use offrozen semen in the dog. Vet Rec 92:6, 1973 Smith FO: Update on freezing canine semen. In Kirk RW (ed): Current Veterinary Therapy IX. Philadelphia, WB Saunders, 1986, p 1243 Smith MS, McDonald LE: Serum levels of luteinizing hormone and progesterone during the estrous cycle, pseudopregnancy and pregnancy in the dog. Endocrinology 94:404, 1974 Tsutsui T: Studies on the reproduction in the dog. VI. Ovulation rate and transuterine migration of the fertilized ova. Jpn J Anim Reprod 21:98, 1975 Tsutsui T: Gamete physiology and timing of ovulation and fertilization in dogs. [Suppl] 39:269, 1989 Tsutsui T, Kawakami E, Murao I, et al: Transport of spermatozoa in the reproductive tract of the bitch: Observations through uterine fistulas. Jpn J Vet Sci 51:560, 1989 Tsutsui T, Shimizu T: Studies on the physiology of reproduction in the dog: IV. On the fertile period of the ovum after ovulation. Jpn J Anim Reprod 21:65, 1975 Tsutsui T, Shimizu T, Ohara N et al: Relationship between the number of sperms and the rate of implantation in bitches inseminated into unilateral uterine horn. Jpn J Vet Sci 51:257, 1989 VanDemark NL, Sharma VD: A preliminary report on preservation of bovine semen at room temperature. J Anim Sci 15:1212, 1956 Van der Holst W, Best AP: Een beschouwing over het meest geschikte tijdstip voor de dekking van de teef. Tijdschr Diergeneesk 101:658, 1976
Address reprint requests to Catharina Linde-Forsberg, DVM, PhD Department of Obstetrics and Gynecology Veterinary Medical Faculty Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden
0195-5616/91 $0.00
+ .20
Achieving Canine Pregnancy by Using Frozen or Chilled Extended Semen Catharina Linde-Forsberg, DVM, PhD*
The use of artificial insemination (AI) in dog breeding has experienced a tremendous increase in popularity over the last few years as breeders become increasingly aware of the fascinating possibilities that it offers, and the results of AI are improving as new knowledge and better techniques for semen preparation, estrous detection, and insemination become available. The most suitable stud can now be chosen even if it is thousands of miles away. The age of the stud may not even be a factor if its owner has been foresighted enough to store its genes in a semen bank. Particularly for the less common breeds, access to imported semen can be crucial in avoiding the adverse effects of inbreeding, thereby keeping the breed healthy and sound. In most countries, the kennel clubs, in one way or another, restrict the right to freeze and store dog semen to a few approved agencies with strict record-keeping practices. In this way, the origin of each semen dose can be determined reliably. Actually, the limiting factor today for making full use of the technique is the number of practitioners specializing in small-animal reproduction who are available to help breeders with advice and inseminations. It is hoped that within the next few years many more colleagues will discover and engage in this rewarding field of work. The factors most important in determining the success of artificial insemination in dogs are
l. Timing of the insemination 2. Semen quality and handling 3. Insemination technique THE PROPER DAY FOR INSEMINATION The bitch is unique among the domestic animals with her long period of heat and receptivity. Much knowledge has been gained during the last *Associate Professor, Department of Obstetrics and Gynaecology, Veterinary Medical Faculty, Swedish University of Agricultural Sciences, Uppsala, Sweden Veterinary Clinics of North America: Small Animal Practice-Yo!. 21, No. 3, May 1991
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10 years regarding her reproductive physiology and endocrinology, and excellent review articles on these subjects have been published. 8 • 9 • 49 The long duration of bitches' heat period creates many practical diagnostic problems, especially because the variation between bitches is very pronounced. The canine proestrus, that is, the time from the first day of observed vaginal hemorrhage until readiness to stand for the male dog, averages 9 days but can be as short as 2 to 3 days or as long as 27 days. 60 The estrus, that is, the period of receptivity, qlso averages 9 days but may be as short as 2 to 3 days or as long as 21 days. 13 Thus, some bitches should be inseminated in the first week of their heat, whereas others should not be inseminated until after day 27. Many breeders, however, still stick to the old rule of thumb that bitches should be mated between days 10 and 14. As a result, bitches with divergent but normal heat periods will not conceive. The breeders often need help to diagnose those extreme heat periods. It is clear that it is not possible to determine the fertile days in the heat period accurately if timing is based on the first day of proestrus. On the other hand, the bitch can become pregnant after a single mating from about 1 day before until about 7 or 8 days after luteinizing hormone (LH) peak. 35 • 60 These assumptions are based on findings that the semen maintains its longevity for about 4 to 7 days, 15• 18 • 58 the LH peak precedes ovulation by 36 to 50 hours, 11 • 50 and the ova undergo maturation divisions for another 2 to 5 days before obtaining the capacity to become fertilized 4 • 31 · 57 and remain alive for 1 to 4 days thereafter. 13· 58 The capacitation time of canine spermatozoa has been estimated to be 7 hours. 41 Thus, theoretically the optimum time for mating would be around day 4 after the LH peak when the ova are shed and most of them have completed maturation division (Fig. 1). Diagnostic Methods Clinical Signs. To determine the optimum days for inseminating the bitch, the clinical signs of vulvar swelling and vaginal discharge and the Optimal mating time
LH-PEAK
!
D
+-----------MATING----------•
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DAYS FROM LH-PEAK Figure 1. Temporal relationship between LH peak, ovulation, maturation of ova, and the hypothetically maximal timespan for successful mating of the bitch. (From Linde C, Karlsson 1: The correlation between the cytology of the vaginal smear and the time of ovulation in the bitch. J Small Anim Pract 25:77, 1984; with permission.)
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behavior of the bitch when teased with a male dog can be used. Usually, the swelling of the vulva has receded somewhat, hemorrhaging has diminished, the discharge is more straw-colored, and, ideally, the bitch has already allowed the male dog to mount her for a couple of days. The owner should be asked to supply these details. During insemination, palpation to determine the size and consistency of the cervix also provides clues as to the stage of estrus, because the cervix responds to changing levels of estradiol in a manner similar to that of the vulva. That is, during proestrus and early estrus it grows large and hard, and during late estrus it diminishes in size and becomes softer. Vaginal Examinations. A quick and simple diagnostic method to determine the best time for insemination is to take a vaginal smear and determine the degree of cellular cornification under a microscope. 7 • 35· 52 This will give the experienced examiner a good indication as to whether or not the bitch is in proper estrus. Because the cellular changes are caused by changes in estradiol, however, no firm conclusions as to whether ovulation has taken place can be made. In the dog, there have not been any reports of specific cellular changes in the vaginal smear being caused by the rise in progesterone following ovulation. For mating or insemination with fresh semen, which is expected to retain its fertilizing capacity in the uterus of the bitch for between 2 and 11 days, 15· 18· 58 the vaginal smear still is very useful. However, when using frozen semen, which has a comparatively short postthaw survival time, more specific methods are required to determine when the ova are shed and ready to be fertilized in the oviduct. 36 The endoscopic appearance of the vaginal vault during different stages of the estrus cycle has been described. 37 • 38 This method may prove very useful for clinicians who have access to an endoscope. Strips for measuring the vaginal glucose content63 or the electrical resistance of the vaginal mucus 29 have been evaluated, but both methods suffer from the same limitations as the vaginal smear: they reflect the effects of estradiol but not of progesterone. Changes in LH and Progesterone During the Estrous Cycle. Changes in LH levels during the estrous cycle of the dog have been reported by several researchers. 5 · 6 • 11 • 12• 45 • 47 • 56 LH levels that are low during proestrus increase 20- to 40-fold during the preovulatory surge at the end of proestrus. The preovulatory rise in LH lasts about 24 to 72 hours and causes ovulations 36 to 50 hours after the LH surge. LH release patterns in the dog, as in other mammalian species, are pulsatile. LH concentrations are 0.2 to 1.2 ng/mL between pulses during anestrus, reach levels of 2 to 25 ng/mL, average 5 ± 1 ng/mL during episodic pulses, and peak at 8 to 50 ng/mL with an average of 20 ng/mL during the preovulatory LH surge. 13· 14 Progesterone levels in the bitch are less than 0.5 ng/mL (25 ng/mL (>78,5 nmol/L). (Fig. 2). Hormone Assays. The most accurate methods available for determining the time of ovulation in the bitch involve measuring peripheral plasma
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PROGESTERONE 25 (78.5)
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ESTRADIOL
20 (63)
~
E
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e Q)
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Estrus
Metestrus
Figure 2. Schematic representation of the hormonal events during the heat period in the bitch and the ideal time for insemination.
levels of LH or progesterone. Recently a rapid radioimmunoassay (RIA) for LH analysis has been developed that will give a result within 3 hours. The results of this method are comparable to those of the much longer standard LH assay. 40 The advantage with the LH assay is that the breeder will know several days in advance when to inseminate the bitch, because the LH peak precedes ovulation (see earlier discussion). The disadvantage is that the LH peak in the dog has a duration of only 1 to 2 days, which necessitates daily blood sampling (for the rapid RIA) or sampling every other day (for the long RIA) and frequent analyses. There are several RIA and ELISA methods available for progesterone analysis. 17• 19• 21 • 32 Most of the commercially available ELISA kits are semiquantitative as they are adapted for use in other species. Thus, they are too unspecific to be of help. Using such kits, one can determine whether the peripheral plasma progesterone is below or above a certain level, usually around 7 ng/mL (22 nmoi/L). Because the shift in color indicating the higher level of progesterone is difficult to discern with the naked eye, a photometer may be needed, and such instruments are expensive. 17 The more quantitative ELISA kits require micropipetting of very minute volumes (20 !J.L) and demand a great deal of routine laboratory practice to give accurate and repeatable results. Thus, they are not as practical as they may seem at first. With a sensitive RIA technique, the progesterone level can be given with an inter- and intra-assay variation of not more than 10% at plasma levels above 3 ng/mL (10 nmol!L). 22 • 32· 39 The assay takes from 6 to 24 hours. With the Coat-A-Count (Diagnostic Products Corp., Los Angeles, California) method the analysis may take no longer than 3 hours. 22 Therefore, the practitioner is advised to establish contact with a good
ACHIEVING PREGNANCY BY USING FROZEN OR CHILLED EXTENDED SEMEN
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referral laboratory for canine progesterone analysis. If the sample is posted immediately after collection, the results should be available over the telephone the following afternoon. When to Inseminate Studies have shown that inseminations with fresh semen performed so early that the progesterone level was still below 5,44 ng/mL (17,1 nmol/L); that is, before ovulation, often may result in pr~gnancy. With frozen-thawed semen, however, which has a shorter postthaw survival time, only one pregnancy resulted from 15 inseminations performed at a progesterone level below 9.6 ng/mL (30 nmol!L). 36 Ideally, the progesterone level should range between 10 and 25 ng/mL (30 and 75 nmol/L), which means that the bitch should be inseminated late in estrus (Fig. 2). The most practical way to effect this is to estimate the correct day with the aid of the details given by the owner of the bitch. Then, the bitch should be brought in for sampling 1 or 2 days before this estimated date. If it is still slightly early, the progesterone probably has gone up from baseline levels, and the proper day can be calculated from the value obtained by the RIA progesterone assay. On the other hand, if the bitch is late and her progesterone already at >25 ng/mL (>78.5 nmol/L), a vaginal smear must be taken to find out whether she is already in metestrus. Because the maximum level of progesterone may be maintained for several weeks, progesterone levels in these cases cannot be used to estimate the time elapsed since ovulation.
SEMEN QUALITY AND HANDLING Semen Collection Canine semen is easily collected by manual massage. Most dogs are very cooperative. If they are timid or feel uncomfortable in the surroundings, it may help if the veterinarian attempting collection is not wearing a white coat, which may bring about unpleasant memories. The collection should take place with the dog standing on the floor. A bitch in heat may be helpful but usually is not necessary. Her presence, however, might result in a higher total number of spermatozoa in the ejaculate. Dogs' ejaculate is distinctly fractionated . The first fraction is watery, emanates from the prostate gland, and usually has a volume of 1 to 5 mL. It takes around 0.5 to 1 minute to complete. The second fraction is milkish white, 1 to 3 mL in volume, and contains the major portion of the spermatozoa. It takes around 1 to 2 minutes to complete. The third fraction comes from the prostate, constitutes the major portion of the ejaculate, and may be up to 30 to 40 mL in the larger breeds. It may take from around 5 minutes to 1 hour to complete. For insemination purposes, only the second, sperm-rich fraction is of interest. A gloved hand is introduced from behind between the hindlegs of the dog and the caudal part of the bulbus glandis is stimulated by manual massage. When semi-erection is achieved, the preputial sheath is slid backwards so that the bulbus glandis is freed and the penis may be turned
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around allowing collection to take place behind the dog. With this technique, the glans penis and the preputial lining may be inspected for abrasions, wounds, or signs of infection. Also, if a bacterial sample is to be collected, contamination by dust or hair from the dog's coat is avoided (Fig. 3). For semen collection, some people use a rubber cone with a tube attached to the end whereas others slip a plastic bag over the dog's penis. With these methods, however, pus from the prepuce may contaminate the semen sample. Moreover, fractioning of the ejaculate is difficult or impossible. Semen also can be collected in a pre-warmed, calibrated glass or plastic vial equipped with a wide opening and a tapering end. By holding the vial in the closed hand, the semen is protected from changes in temperature. During the pelvic thrusting, the vial should be held at a distance from the penis to avoid trauma and possible hemorrhage. If the dog is reluctant to ejaculate, the thumb of the hand holding the vial can
Figure 3. Semen collection technique in the dog. The penis is flexed backwards. and collection takes place behind the dog.
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be introduced into the fossa of the glans and pulsatile pressure can be applied to the urethral process against the wall of the vial (Fig. 4): Most dogs like this, although some may react adversely. If the first, watery semen fraction is large or fouled by urine or blood, the collecting vial should be emptied by rapidly turning it upside-down and the collection continued without interruption and without distracting the dog. If the semen is very dilute or if the dog has prostatitis, the sample should be gently centrifuged for 5 minutes at 300 G to 700 G24 and the supernatant seminal plasma discarded. Then, the semen is diluted to the desired density and volume with an extender and, in the case of infection, with antibiotics. In this way, both semen quality and longevity can be substantially enhanced. Semen Quality No comprehensive minimum requirements for dog semen to be fertile are available, largely because the reproductive pattern of this species and the way dogs are kept and bred makes them unsuitable for systematic studies. Most published studies therefore are based on clinical material and on inseminations of patients. It should be kept in mind that fertility is not an ali-or-none trait. Poor semen quality influences mean pregnancy rates and mean numbers of puppies based on a large number of matings. But on the individual level, poor quality semen may still result in pregnancy and normal litter size. Most researchers agree that the total number of spermatozoa per insemination should be between 150 to 200 X 106 • 2 • 3• 43 On the other hand, good pregnancy results have been achieved using as few as 20 X 106 fresh spermatozoa deposited surgically at the tip of the uterine horn. 61 This is in agreement with findings by Fougner and Forsberg27 who achieved normal pregnancy and litter size in vixens inseminated into the uterus with as few
Figure 4. Applying pressure on the processus urethralis against the wall of the collecting vial may stimulate reluctant dogs to ejaculate.
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as 20 X 106 fresh spermatozoa. No comparable studies have been made with frozen dog semen. The proportion of spermatozoa with primary and secondary defects should not exceed 30% to 40%. 20· 25 • 33 On the other hand, some found that the proportion of spermatozoa judged to be normal ranged between 0% to 96% among 167 normal dogs. However, the criterion for normality in this study was that the dogs had sired a litter during the last 12 months. 44 A German Shepherd that was used for approxi!,llately 100 matings per year had 60% defective spermatozoa (excluding distal protoplasmic droplets) and a total sperm count of 1.300 x 106 . About half of the bitches mated to this dog did conceive (Linde-Forsberg, unpublished data). Again, the relative significance of different types of defects has been studied very little. It generally is agreed that distal protoplasmic droplets lack significance, although they may be a signal of an ongoing disturbance. Motility is said to be an important parameter in judging canine semen quality and should exceed 70% in a normal semen sample. When dealing with insemination in dogs, semen that has been extended and possibly frozen usually is used. Because some semen extenders reduce motility, it is difficult or impossible to make a judgement unless the composition of the extender is known . Pregnancy was obtained and eight puppies were eventually produced with imported fresh extended semen that had a motility of only 10%. 36 The composition of the extender in this case was unknown. With frozen semen, pregnancies were obtained with semen with a postthaw motility between 20% to 30%. 36 When judging sperm motility, the semen sample should always be allowed to reach room-temperature (37°C is preferred). A good indication of its quality is also obtained by looking at its resistance, that is, the length of time that it retains motility. Morton and Bruce44 compared the longevities of fresh semen stored at 4°C without extender with a Tris-extender and frozen-thawed semen with an extender. The results clearly show that the frozen-thawed fresh semen has the shortest survival time and that the best results, under these conditions, were achieved for fresh-extended semen. 44 Handling of Fresh Semen Semen handling is crucial to achieve good results. Canine semen may survive cooling to 4°C for several days. 44 It is, however, important that the cooling process is gradual (30 to 60 minutes). The most important step in the cooling procedure is that from body temperature to room temperature. This may increase the longevity of the spermatozoa sixfold. Unless the semen can be cooled under well-controlled conditions, it is better to store it at room temperature. In other words, it is better not to chill the semen than to chill it too fast or too much. If the semen is stored with ice, it is imperative that the semen vial is well-insulated so that there is no direct contact with the ice cubes; otherwise, the temperature can drop so low that the spermatozoal membranes disintegrate, leading to enzyme leakage. Although such spermatozoa may show good progressive motility when examined under a microscope after warming them up, they have no fertilizing capacity. At least 15 minutes should be allowed for the subsequent warming of the semen to room temperature before use. Always try to avoid temperature fluctuations.
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When semen is to be·chilled and shipped it should always be extended. The extender helps to keep the spermatozoal membranes from being harmed by changes in temperature and shaking during transport while also providing energy and stabilizing the pH. Morton and Bruce44 clearly showed that the survival time of spermatozoa in extended semen is considerably higher than that of undiluted semen stored at 4°C. Progressive motility after 24 hours was 91.7% for undiluted semen and 98% for diluted semen. After 48 hours, the motility of the undiluted semen was only 36.7% whereas that of the diluted semen was 90%. After 72 hours, the motility of the undiluted semen was down to 18.3%, whereas after 96 hours, the diluted semen still showed 80% motility. 44 Seminal Extenders There are many types of extenders for canine semen. Some of the first described consisted of pasteurized milk: 30 The Illinois Variable Temperature (1. V. T.) extender, 61 and 1.16% sodium citrate dihydrate, 0. 75% glycine, and 1% glucose with 20% egg yolk (v/v). 26 Today, one of the most commonly used extenders is a Tris-citrate buffer with 20% egg yolk and antibiotics. 16 The author's favorite extender for fresh semen consists of 80% homogenized and pasteurized cream (12% fat) and 20% (v/v) egg yolk. One mg of dihydrostreptomycin and 1000 IU of benzyl penicillin are added per mL of final solution. For an average ejaculate consisting of 1 to 2 mL of spermdense, second-fraction semen, 4 mL of pasteurized cream is mixed with 1 mL of egg yolk. The egg yolk should be carefully separated from the white by placing the former on a filter paper and gently rolling it around until the white has come off. Use a 2-cc disposable syringe to measure the correct volume of egg yolk. Before mixing the extender and the semen, make sure that the two solutions are at the same temperature. The dilution rate usually ranges from 1:3 to 1:5. The final volume of extended semen should not exceed 5 to 10 mL per insemination dose. Chilling of Fresh Semen
If the semen is to be chilled, place the semen vial (preferably a sterile plastic tube with a screw cap that will not break during transport [e.g., a Nunc-tube]) in a glass filled with room-temperature water before putting it in the refrigerator. Leave the tube in the refrigerator for 30 to 60 minutes until the temperature has dropped to the desired level, remembering not to chill it too fast or to too low a level. For most transport distances, a temperature between 10° to 15°C should be sufficient. At the same time, put the thermos bottle filled with moistened cotton wool as shock absorbant in the refrigerator so that it will be the same temperature as the semen sample. Semen prepared in this manner will maintain its fertilizing capacity for at least 12 to 24 hours and usually longer. Frozen Semen
If dog semen is to be frozen, the extender must also contain glycerol. It is not within the scope of this article to describe freezing techniques,
because most practitioners are not in a position to freeze semen. For
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those interested, several papers and review articles have been published. 2. 10, 44, 53, 54, ss Thawing of Frozen Semen. Frozen semen should always be thawed as recommended by the person freezing it, because the method of choice depends on the method of freezing. Rapid freezing requires rapid thawing and vice versa. This information has to be supplied by the freezing agency. The semen can be frozen either in pellets or in straws (usually 0.5 mL but occasionally 0.25 mL). The pelleted semen is always thawed in a thawing medium, usually saline or citrate, at 37°C. Semen frozen in straws may be thawed with or without thawing media. The 0.5-mL straws are thawed in a 37°C water bath for 15 seconds and a thawing medium is used (Govette, personal communication), or bathed for 60 seconds, 43 or in a water bath of 75°C for 6.5 seconds, 1. 2 or for 12 seconds. 46 For mini-straws (0.25 mL), the thawing time should be reduced by approximately one half. Fresh or Frozen Semen? Most canine inseminations today are performed with fresh semen. It is easy to handle and ship, and the insemination technique used is relatively simple. Fresh, usually extended and chilled, canine semen may, if of originally good quality, retain its fertilizing capacity for at least 12 to 24 hours, usually considerably longer. This means that it can be used for most national shipments, and for many international ones as well. The author, who practices in Europe, considers the advantages of using fresh semen to outweigh the disadvantages in almost every case, especially because results thus far are better than for frozen semen. A study of the results of 470 inseminations showed that both pregnancy rate and litter size are higher for bitches receiving fresh semen. 36 The pregnancy rate was 83.8% with good quality fresh, chilled extended semen compared with 69.3% with good quality frozen semen. Litter size was estimated to be 23.3% smaller in the group of bitches receiving frozen semen, or 29.7% on a within-breed basis. 36 In a smaller number of inseminations, Farstad 23 obtained a pregnancy rate of 84% with fresh and 67% with frozen semen but no reduction in litter size with frozen semen. 23 Fresh semen may be shipped in an ordinary thermos bottle, preferably one with a wide opening. It may be forwarded by air as an ordinary parcel at a modest rate. The transport thermos is of no great value and need not be returned to the sender. (It obviously can be kept by the buyer to be used for its intended purpose.) The insemination can be made in the cranial vagina, which is technically quite easy for a trained inseminator. The disadvantage with fresh semen is that everything has to be arranged on the day most suitable for the bitch. Usually at least four people have to be available at a certain date. The owner of the stud dog has to contact his or her veterinarian for the semen collection and book a convenient flight. At the same time, the owner of the bitch has to arrange with his or her veterinarian to have the insemination done. Thus, the potential for encountering problems is high, especially if the date in question is a holiday. The advantages with frozen semen are that it can be shipped at a time convenient for all parties and it can be available when it is needed. Moreover, only two people have to be available on the chosen day, the
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bitch owner and the inseminating veterinarian. Also, if the semen is shipped a long distance or even imported, many doses can be sent in a single shipment to be used when desired. The semen banking option certainly will prove to be of exceptional value in dog breeding because the semen may be stored practically forever. The disadvantages of frozen semen have already been mentioned: Only a limited number of technicians have mastered the freezing technique, and the kennel clubs only allow it to be performed .by a few people and agencies. Frozen semen usually has to be shipped as dangerous goods with all the extra charges that this entails. This, however, may be avoided by using one of the modern dry-shippers. These liquid nitrogen containers absorb the nitrogen into their walls, thereby preventing spillage even if the container is turned on its side or upside-down. This also means that the hazards of ruining the semen during transport are minimized. For those involved in this business, an investment in this modern equipment is highly recommended. The container is expensive and usually of no further use to the buyer of the semen and is thus returned to the shipper, which entails some extra costs. The insemination technique using frozen semen is considerably more difficult than that used for fresh semen inseminations (see later discussion). Depositing frozen semen in the cranial vagina generally results in a very poor pregnancy rate. 2• 3 • 36 In conclusion, the use of fresh-extended and chilled semen is usually the method of choice because it is easy, less expensive, and gives a better result. With good planning, commencing as soon as the bitch comes into heat, the problem of organizing the collection and shipment of semen and the insemination should not be too difficult to overcome. In most cases, the longevity of the semen is good enough to allow for transport times of at least 12 to 24 hours. Frozen semen should be recommended when the transport times are too long, when semen for more than one bitch is requested, when the people involved are not available on the best day for insemination of the particular bitch, and, obviously, when semen is to be banked for future use. INSEMINATION TECHNIQUES The current methods for inseminating bitches include vaginal deposition, transcervical intrauterine deposition, surgical intrauterine deposition, and intrauterine insemination by laparoscopy. In many countries, the latter two methods are considered to be unethical and they are not discussed further in this article. Insemination Catheters For vaginal inseminations, a 45 x 0.5 em disposable plastic catheter is used (originally for uterine infusions in the bovine). For transcervical intrauterine inseminations, a 20- to 50-em-long steel catheter with a 0.5mm to 1-mm diameter tip is used together with a protecting nylon sheath (Fig. 5). The latter is a Norwegian construction adapted from a model used
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Figure 5. Insemination catheters. Top, the plastic catheter used for deep vaginal inseminations in the bitch. Below, three sizes of the Norwegian catheter for intrauterine inseminations. A 5- to 10-cc syringe should be used for the semen.
for inseminations of farmed foxes. 2 Usually, three different lengths of the catheter are sufficient for insemination of most breeds. There also is a modified version of the latter catheter that has an outer metal sheath with a loop at the end intended to fit around, and thus fixate, the portio vaginalis of the cervix. 28 The advantage of this model is that once the cervix is fixed in the loop, it may be rotated easily to facilitate palpation and introduction of the inner catheter through the cervical canal. The disadvantage is that roughly 10 different sizes of loops are necessary to cover the variation in size of the portio vaginalis among dog breeds. A French company is marketing a flexible vaginal catheter with an inflatable cuff intended to be left in situ for some time after insemination. 42 Thus far, there are no published data indicating that results with this equipment are superior to those obtained with the standard deep-vaginal insemination of fresh semen or the transcervical insemination of frozen semen followed by holding the hindquarters of the bitch elevated for 10 minutes. Abdominal Palpation The clinician conducting canine inseminations should learn to locate the cervix of the bitch through abdominal palpation in order to be able to deposit the semen in the correct place . Correct placement is as crucial for vaginal inse minations as for intrauterine inseminations. From the size and consistency of the cervix the clinician can also tell whether the bitch is at the right stage of the estrous cycle (see earlier discussion). Because the uretheral opening of the bitch is located at the brim of the pelvic floor, it is surprisingly easy for the catheter to be unintentionally introduced into the urinary bladder. If the bladder is void of urine or if the syringe containing the semen is applied to the end of the catheter, no urine is seen in the catheter to indicate its position in the bladder. Apart from the hazard of perforating the bladder with the catheter, it is obvious that no pregnancy
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will follow. Thus, the correct position of the tip of the catheter should always be checked by palpation before depositing the semen dose. Vaginal Insemination The bitch to be palpated or inseminated should have an empty stomach to facilitate the procedure. To locate the cervix, the plastic catheter is introduced into the vagina of the bitch. No speculum is needed. The angle of introduction is either steeply upward until the pelvic brim has been passed, or the vulva is elevated until it is just below the anus (as the bitch does when inviting the male dog) and the catheter is introduced straight ahead. The vagina then slopes slightly forward and downward (Fig. 6). When the tip of the catheter is in front of the pelvic floor, it is palpated to help orientation. By elevating the distal end of the catheter, the tip is lowered closer to the abdominal wall and thus becomes more accessible to palpation. The catheter is carefully introduced until it reaches the narrow, cranial portion of the vagina created by the dorsal, median postcervical fold 51 (see Fig. 6). In some bitches, especially maiden ones or ones of small breeds, it is not possible to pass the catheter all the way through to the cervical fornix. The cranial vagina can be felt as a 1- to 2-cm-long, firm structure that ends at the cervix, which in a bitch in estrus is a 0.5- to 1.5cm hard, rounded to ovoid structure that is freely movable. The corpus uteri and the uterine horns can be felt in front of the cervix. To palpate them, work forward with the aid of the catheter by lowering its tip, closing the tip of the thumb against that of the index finger above the catheter; and then lifting its tip so that the cervix or the uterine horns are forced
Figure 6. Colpogram of an estrous bitch clearly outlines the narrow cranial part of the vagina and the fornix surrounding the estrogen stimulated cervix (on the left). (Courtesy of Anne-Sofie Lagerstedt, DVM.)
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upward between the fingers (Fig. 7). Their size and consistency then become evident. This method of palpating the uterus also is very useful in examining a bitch with suspected endometritis or pyometra and for early pregnancy detection from 3 to 5 weeks after mating. Because the plastic catheter is much too wide to be able to pass through the normal cervical canal, there is no risk involved for the fetuses. If the catheter is in the urinary bladder, the thick, cranial part of the vagina and the cervix are palpable above the catheter. In addition, because the walls of the urinary bladder usually are much thinner than those of the vagina, the tip of the catheter stands out more clearly. Even though the urine usually is sterile, it is recommended that the catheter be exchanged for a new one if the bladder has been mistakenly catheterized. Intrauterine Insemination In transcervical intrauterine insemination, the steel catheter is introduced into the vagina, which is protected by the nylon sheath. Once the pelvic floor is passed, the cranial end of the nylon sheath is palpated as described earlier. It usually is too wide to be introduced into the cranial, narrow part of the vagina. Before its introduction, a mark is made on the distal part of the steel catheter to indicate when its tip is in front of the nylon sheath. The sheath is palpated, and the cervix usually is found 1 to 2 em in front and slightly above the sheath if the tip of the catheter has been lowered closer to the abdominal wall. The steel catheter then is introduced through the nylon sheath all the way into the fornix, that is, below the portio vaginalis of the cervix. The cervix is fixed through the
Figure 7. Abdominal palpation in a bitch with the aid of a vaginal catheter. By lowering the tip of the catheter (on the left}, the cranial vagina, cervix, and caudal part of the uterine horns become accessible for palpation.
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abdominal walls between the thumb and the index finger and, by a slightly downward traction at the corpus uteri, it is tilted so that the angle of the cervical canal is changed from between 45° to 60° to a more horizontal angle (Fig. 8). The tip of the catheter is then moved, using very small movements caudally and touching against the cervix in search of the opening of the cervical canal. The sensation when the opening is found can best be described as the sensation of touching cartilage, that is, "crispy." Once the opening has been found, fix the catheter anel start working the cervix against the catheter. The cervical canal may be some 5 to 10 mm long, and is not always straight. Thus, it often has to be slightly forced onto the catheter. In most bitches, the tip of the catheter easily can be felt in front of the cervix in the corpus uteri. In some bitches, however, the sensation is not as distinct. For training purposes, saline can be infused to check the correct position of the catheter in the uterus of the bitch. If the catheter is in the right position, the fluid can easily be infused, and if air is present in the catheter or syringe, as is often the case, a characteristic, bubbling sound may be heard when introducing the fluid through the cervix. If, on the other hand, the catheter still is in the vagina, there will be a backflow between the catheter and the nylon sheath. Another way to control the position of the tip of the catheter during training is to infuse 60% U rographin or Amipaque and to radiograph the bitch. The bitch should be held with elevated hindquarters for at least 10
A
Figure 8. Sagittal section of the canine cranial vagina and cervix with the Norwegian intrauterine insemination catheter in position. A, The cervical canal is at an angle of approximately 45° to 60° to the cranial vagina. B, To facilitate catheterization, the cervix is fixed between the thumb and index finger by abdominal palpation and tilted in a more horizontal position.
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minutes after insemination with fresh or frozen semen to help the uterine transport of spermatozoa toward the oviducts34 (Fig. 9). Studying bitches with experimental uterine fistulas, Tsutsui59 found semen at the tip of the uterine horn about 30 seconds to 1 minute after a natural mating and about 30 seconds to 2 minutes after vaginal insemination when the bitch was held with elevated hindquarters. When the bitch was inseminated standing horizontally, the transport of spermatozoa was greatly hindered. When fresh semen is inseminated, the hindquarters of the bitch are elevated with the catheter in position in the cranial vagina. The bitch should be held so that the catheter is in a vertical position before infusing the semen (Fig. 9). After deposition of the semen, the catheter is withdrawn, and the vulva is feathered to induce the bitch to flag her tail and to stimulate uterine contractions, which supposedly facilitate sperm transport. The frozen-thawed semen is introduced with the bitch standing in the normal position, and the cervix fixed between the fingers. After deposition of the semen, the hindquarters are elevated. The catheter is then withdrawn, and the bitch is held as described earlier. Sedation is usually not
Figure 9. It is important to hold the bitch with its hindquarters elevated after deposition of the semen. The catheter (left in to demonstrate the desired angle) is removed and the perivulvar region feathered to improve uterine transport of spermatozoa.
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needed; on the contrary, most bitches in estrus like this type of handling. Remember to instruct the owner that the bitch should have an empty stomach to facilitate palpation. Insemination Volume Because the uterine lumen in the bitch is quite small, insemination volume should not exceed 5 to 10 mL. Diluting the semen more results in a back-flow of spermatozoa as soon as the ~itch is back on all four feet again. How Many Times Should the Bitch Be Inseminated? Repeated inseminations with either fresh or frozen semen have been reported to result in higher pregnancy rates than single inseminations. 24 • 36 This is probably a result of the extended period of ovulation and maturation of the ova in the bitch. If for practical or economic reasons only one insemination can be made, emphasis should be placed on making sure that the bitch is inseminated at the optimum time, rather late in estrus.
REFERENCES 1. Aamdal J, Andersen K: Fast thawing of semen frozen in straws. Zuchthyg 3:22, 1968 2. Andersen K: Insemination with frozen dog semen based on a new insemination technique. Zuchthyg 10:1, 1975 3. Andersen K: Artificial insemination and storage of canine semen. In Morrow DA (ed): Current Therapy in Theriogenology: Diagnosis, Treatment and Prevention of Reproductive Diseases in Animals. Philadelphia, WB Saunders, 1980, p 661 4. Andersen AC, Simpson ME: The Ovary and Reproductive Cycle of the Dog (Beagle). Los Altos, Geron-X Press, 1973 5. Chakraborty PK: Reproductive hormone concentrations during estrus, pregnancy, and pseudopregnancy in the Labrador bitch. Theriogenology 27:827, 1987 6. Chakraborty PK, Panko WB, Fletcher WS: Serum hormone concentrations and their relationships to sexual behavior at the first and second estrous cycles of the Labrador bitch. Bioi Reprod 22:227, 1980 7. Christie DW, Bailey JB, Bell ET: Classification of cell types in vaginal smears during the canine estrus cycle. Br Vet J 128:301, 1972 8. Concannon PW: Clinical and endocrine correlates of canine ovarian cycles and pregnancy. In Kirk RW (ed): Current Veterinary Therapy IX. Philadelphia, WB Saunders, 1986, p 1214 9. Concannon PW: Canine physiology of reproduction. In Burke TJ (ed): Small Animal Reproduction and Infertility. A Clinical Approach to Diagnosis and Treatment. Philadelphia, Lea & Febiger, 1986, p 23 10. Concannon PW, Battista M: Canine semen freezing and artificial insemination. In Kirk RW: Current Veterinary Therapy X: Small Animal Practice. Philadelphia, WB Saunders, 1989, p 1247 11. Concannon PW, Hansel W, McEntee K: Changes in LH, progesterone and sexual behavior associated with preovulatory luteinization in the bitch. Bioi Reprod 17:604, 1977 12. Concannon PW, Hansel W, Visek WJ: The ovarian cycle of the bitch: Plasma estrogen, LH and progesterone. Bioi Reprod 13:112, 1975 13. Concannon PW, McCann JP, Temple M: Biology and endocrinology of ovulation, pregnancy and parturition in the dog. J Reprod Fertil [Suppl] 39:3, 1989 14. Concannon PW, Whaley S, Anderson SP: Increased LH pulse frequency associated with termination of anestrus during the ovarian cycle of the dog [abstract]. Bioi Reprod 34 (Suppl):ll9, 1986
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15. Concannon PW, Whaley S, Lein D et al: Canine gestation length: Variation related to time of mating and fertile life of sperm. Am J Vet Res 44:1819, 1983 16. Davis JS, Bratton RW, Foote RH: Livability of bovine spermatozoa at 5°, -25° and -85° Cin Tris-buffered and citrate-buffered yolk-glycerol extenders. J Dairy Sci 46:57, 1963 17. Dee J, Forchhammer S: Progesteronmaling hos hund. Fastsaettelse af parringstidspunkt. Dansk VetTidsskr 71:425, 1988 18. Doak RL, Hall A, Dale HE: Longevity of spermatozoa in the reproductive tract of the bitch. J Reprod Fertil 13:51, 1967 19. Eckersall PD, Harvey MJA: The use of a bovine plasma progesterone ELISA kit to measure progesterone in equine, ovine and canine-plasmas. Vet Rec 3:5, 1987 20. England GCW, Allen WE: Seminal characteristics and fertility in dogs. Vet Rec 124:399, 1989 21. England GCW, Allen WE, Porter DJ: A comparison of radioimmunoassay with quantitative and qualitative enzyme-linked immunoassay for plasma progestogen detection in bitches. Vet Rec 124:107, 1989 22. Epelu-Opio J, Madej A: Direct measurement of progesterone in plasma and milk by a simple solid-phase radioimmunoassay. Vet Clin Pathol17:26, 1988 23. Farstad W: Bitch fertility after natural mating and after artificial insemination with fresh or frozen semen. J Small Anim Pract 25:561, 1984 24. Farstad W, Andersen-Berg K: Factors influencing the success rate of artificial insemination with frozen semen in the dog. J Reprod Fertil [Suppl] 39:289, 1989 25. Feldman EC, Nelson RW: Disorders of the canine male reproductive tract. In Canine and Feline Endocrinology and Reproduction. Philadelphia, WB Saunders, 1987, p 481 26. Foote RH, Leonard EP: The influence of pH, osmotic pressure, glycine and glycerol on the survival of dog sperm in buffered yolk extenders. Cornell Vet 54:78, 1964 27. Fougner JA, Forsberg M: Effect of different sperm numbers on fertility after artificial insemination of foxes . Acta Vet Scand 28:403, 1987 28. Funkquist B, Lagerstedt A-S, Linde C, et al: Hysterography in the bitch. Vet Radio! 26:12, 1985 29. Giinzel A, Koivisto P, Fougner J: Electrical resistance of vaginal secretion in the bitch. Theriogenology 25:559, 1986 30. Harrop AE: Artificial insemination of a bitch with preserved semen. Br Vet J 110:424, 1954 31. Holst PA, Phemister RD: The prenatal development of the dog: Preimplantation events. Bioi Reprod 5:194, 1971 32. Kindahl H, Edqvist L-E, Granstrom E et al: The release of prostaglandin F 2a as reflected by 15-keto-13,14-dihydroprostaglandin F 2a in the peripheral circulation during normal luteolysis in heifers. Prostaglandins 11:871, 1976 33. Larsen RE: Evaluation of fertility problems in the male dog. Vet Clin North Am 7:735, 1979 34. Linde C: Transport of radiopaque fluid into the uterus after vaginal deposition in the oestrous bitch. Acta Vet Scand 19:463, 1978 35. Linde C, Karlsson I: The correlation between the cytology of the vaginal smear and the time of ovulation in the bitch. J Small Anim Pract 25:77, 1984 36. Linde-Forsberg C, Forsberg M: Fertility in dogs in relation to semen quality and the time and site of insemination with fresh and frozen semen. J Reprod Fertil [Suppl] 39:299, 1989 37. Lindsay FEF: The normal endoscopic appearance of the caudal reproductive tract of the cyclic and non-cyclic bitch: Post-uterine endoscopy. J Small Anim Pract 24:1, 1983 38. Lindsay FEF, Concannon PW: Normal canine vaginoscopy. In Burke TJ (ed): Small Animal Reproduction and Infertility. A Clinical Approach to Diagnosis and Treatment. Philadelphia, Lea & Febiger, 1986, p 112 39. Madej A, Kindahl H, Nydal C, et al: Hormonal changes associated with induced late abortions in the mare. J Reprod Fertil [Suppl] 35:479, 1987 40. Madej A, Linde-Forsberg C, Garnum F: A rapid radioimmunoassay for determination of LH in dogs [abstract]. J Reprod Fertil [Suppl] 39:329, 1989 41. Mahi CA, Yanagamachi R: Maturation and sperm penetration of canine oocytes in vitro. J Exp Zool196:189, 1976 42. Mialot J-P, Dumon C, Cassou B: Artificial insemination in bitches: Introduction of fresh
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semen with the Osiris flexible gun. Practique Medicale et Chirurgicale de l'Animal de Compagnie 20:213, 1985 Morton DB: Artificial insemination with frozen semen in the dog: Principles of 'DNA fingerprinting.' In Jones DE, Joshua JO (eds): Reproductive Clinical Problems in the Dog. London, Wright, 1988, p 169 Morton DB, Bruce SG: Semen evaluation, cryopreservation and factors relevant to the use of frozen semen in dogs. J Reprod Fertil [Suppl] 39:311, 1989 Nett TM, Akbar AM, Phemister RD, et al: Levels of luteinizing hormone, estradiol and progesterone in serum during the estrous cycle and pregnancy in the Beagle bitch. Proc Soc Exp Bioi Med 148:134, 1975 Olar TT: Cryopreservation of dog spermatozoa [dissertation]. Fort Collins, CO, Colorado State University, 1984 Olson PN, Bowen RA, Behrendt M, et al: Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus and estrus. Bioi Reprod 27:1196, 1982 Olson PN, Bowen RA, Behrendt M, et al: Concentrations of progesterone and luteinizing hormone in the serum of diestrous bitches before and after hysterectomy. Am J Vet Res 45:149, 1984 Olson PN, Nett TM: Reproductive endocrinology and physiology of the bitch. In Morrow DA: Current Therapy in Theriogenology 2. Philadelphia, WB Saunders, 1986, p 453 Phemister RD, Holst PA, Spano JS, et al: Time of ovulation in the Beagle bitch. Bioi Reprod 8:74, 1973 Pineda MH, Kainer RA, Faulkner LC: Dorsal median postcervical fold in the canine vagina. ,Am J Vet Res 34:1487, 1973 Roszel JF: Genital cytology of the bitch. Scope XIX 1:1, 1975 Seager SWJ: Successful pregnancies utilizing frozen dog semen. AI Digest 17:26, 1969 Seager SWJ, Fletcher WS: Progress on the use offrozen semen in the dog. Vet Rec 92:6, 1973 Smith FO: Update on freezing canine semen. In Kirk RW (ed): Current Veterinary Therapy IX. Philadelphia, WB Saunders, 1986, p 1243 Smith MS, McDonald LE: Serum levels of luteinizing hormone and progesterone during the estrous cycle, pseudopregnancy and pregnancy in the dog. Endocrinology 94:404, 1974 Tsutsui T: Studies on the reproduction in the dog. VI. Ovulation rate and transuterine migration of the fertilized ova. Jpn J Anim Reprod 21:98, 1975 Tsutsui T: Gamete physiology and timing of ovulation and fertilization in dogs. [Suppl] 39:269, 1989 Tsutsui T, Kawakami E, Murao I, et al: Transport of spermatozoa in the reproductive tract of the bitch: Observations through uterine fistulas. Jpn J Vet Sci 51:560, 1989 Tsutsui T, Shimizu T: Studies on the physiology of reproduction in the dog: IV. On the fertile period of the ovum after ovulation. Jpn J Anim Reprod 21:65, 1975 Tsutsui T, Shimizu T, Ohara N et al: Relationship between the number of sperms and the rate of implantation in bitches inseminated into unilateral uterine horn. Jpn J Vet Sci 51:257, 1989 VanDemark NL, Sharma VD: A preliminary report on preservation of bovine semen at room temperature. J Anim Sci 15:1212, 1956 Van der Holst W, Best AP: Een beschouwing over het meest geschikte tijdstip voor de dekking van de teef. Tijdschr Diergeneesk 101:658, 1976
Address reprint requests to Catharina Linde-Forsberg, DVM, PhD Department of Obstetrics and Gynecology Veterinary Medical Faculty Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden
