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Modern breeding management in dogs

When it comes to tools that can help you monitor the best time for breeding, mixing the traditional with the new may be just the right prescription. Here's a guide to what you need for successful breeding management.

BEVERLY J. PURSWELL, DVM, PhD, DACT

NIKOLA A. PARKER, DVM, MS, DACT

Department of Large Animal Clinical Sciences

Virginia-Maryland Regional College of Veterinary Medicine Virginia Tech Blacksburg,

VA 24061 SEPTEMBER

September 2000 Veterinary Medicine

BREEDING MANAGEMENT is an important component of successful planned reproduction in dogs. Mismanagement accounts for many of the breeding problems you see in practice. Because of the wide variation seen in normal canine reproductive cycles, it is not surprising that management issues confuse breeders and veterinarians alike. This article describes the tools that can help you assist dog breeders in obtaining pregnancies in their bitches.

Routine breeding management

Laboratory tests

Brucella canis testing should always be part of a prebreeding workup in both male and female dogs. It is advisable to require Brucella species screening in all outside bitches coming to a stud dog. Bitches are usually asymptomatic carriers of B. canis, and late?term abortion is often the first sign of infection. You should periodically screen a stud dog that is routinely servicing outside bitches even though it is unlikely that a male dog would be infected with B. cams without showing clinical signs, such as epididymo?orchitis.

The rapid slide agglutination test is an excellent in?house screening test (D?Tec CB Canine Brucellosis Antibody Test?Synbiotics). If the slide test yields a positive result, perform additional testing to rule out a false positive result. A negative agar gel immunodiffusion test result rules out a false positive result.' The agar gel immunodiffusion assay tests for cytoplasmic and cell surface antigens, effectively differentiating between true positive and false positive results.

Stud dog owners often request a vaginal culture before an outside bitch is bred. But without clinical signs or a history of reproductive dysfunction, vaginal cultures are usually not indicated. Bitches have normal vaginal flora, which has been described. In one study of normal reproducing brood bitches, 98% of the bitches had positive vaginal culture results for Pasteurella multocida, 89% for (3%hemolytic streptococci, 84% for Escherichia coh, 67% for Pasteurella species, 59% for Mycoplasma species, 55% for Streptococcus species, 44% for enterococci, 40% for Coryneform species, 33% for Staphylococcus intermedius, 25% for Proteus mirabilis, 22% for coagulasenegative staphylococci, and 10% for Pseudomonas species. Similar results were found in cultures from the prepuces of normal stud dogs.

Requiring routine prebreeding vaginal cultures from bitches not only wastes time and money but may lead to inappropriate antimicrobial therapy. One study showed that using antimicrobial drugs in healthy bitches promoted the growth of opportunistic pathogens in the vagina such as E. coli and Mycoplasma species . Some of the normal bitches in this study developed vulvar discharges during antimicrobial therapy.

If an owner still requests a vaginal culture, you must carefully interpret the culture results. Use the findings of a thorough history and physical examination and vaginal cytology to help interpret a positive result. Obtain vaginal cultures with a guarded swab, preferably by using a sterile speculum. The vulva and vestibule are heavily contaminated with bacteria, so avoid them when culturing the vagina.

Scheduling the breeding

It is not unusual for the vulva to be swollen several days before the onset of bloody discharge or for bitches to attract male dogs several weeks before the onset of bloody discharge. Serum estrogen concentrations are quite high for two months before the onset of bloody discharge. Male dogs can detect the pheromones associated with elevations in estrogen before the onset of clinically apparent proestrus.

After the onset of proestrus, a bitch should be brought to a male dog for introductions and detection of standing behavior. It is best to take the bitch to the dog because of territorial considerations. Bitches will feel less dominant outside their home territory, and male dogs must be more dominant than bitches for the bitches to allow breeding. Dominant (alpha) bitches will not allow breeding by less dominant (beta) males. Once standing behavior has begun, breeding should occur every two or three days until the bitch will no longer stand for the dog. On average, bitches will begin to stand on Day 9 or 10 and will quit standing on Day 16 or 17. (For the sake of consistency, in this discussion Day 1 refers to the first day of bloody discharge.) A history of a bitch's previous estrous cycles will help detect the normal variations seen among bitches. Proestrus normally ranges from 2 to 21 days, and estrus also normally ranges from 2 to 21 days. So flexibility is required to accommodate these wide variations.

Routine breeding management will suffice in most breeding situations. If a normal bitch is bred at the correct time to a fertile dog, the bitch should get pregnant.

Aids to breeding management

Vaginal cytology Practitioners must understand why cytologic changes occur and what the changes indicate. The vagina is lined with squamous epithelium that responds to estrogen by increasing in thickness. Normally, the vaginal epithelium is only a few cell layers thick and is susceptible to injury by even the lightest touch. This is demonstrated by the fact that petechial hemorrhages are common when vaginoscopy is performed at any time other than proestrus and estrus. When under the influence of estrogen, the vaginal epithelium thickens to 20 to 40 cell layers. This thickening protects the vagina during copulation. As the vaginal epithelium thickens, exfoliative vaginal cytology shows the change from noncornified to cornified epithelial cells. When cornified epithelial cells predominate, the bitch is either in proestrus or estrus.

Vaginal cytology is not exact enough to determine the onset of ovulation or the optimal time of breeding. Red blood cells are present during proestrus and tend to disappear during estrus, but some normal bitches bleed profusely throughout both proestrus and estrus. White blood cells tend to disappear during estrus. The thickened vaginal epithelium blocks neutrophil migration to the vaginal lumen. Rarely, a normal bitch will continue to display neutrophils in vaginal cytology during estrus. The value of vaginal cytology is often overestimated in breeding management. But one absolute finding in vaginal cytology is the onset of diestrus. The vaginal cytology changes abruptly and dramatically over 24 to 48 hours from a predominantly cornified pattern to a predominantly noncornified pattern. Neutrophils are present in large numbers. The first day of this dramatic cytologic change is referred to as Diestrus Day 1 or D1. This information is valuable because it can be used to predict gestation length. Bitches typically whelp 56 or 57 days after the onset of cytologic diestrus.

Vaginoscopy

Vaginoscopy is a more accurate diagnostic breeding management tool in bitches than vaginal cytology. Under the influence of estrogen, the vaginal folds become swollen, moist, and pink. As a bitch progresses through proestrus and into estrus, these folds begin to lose their swollen appearance and become wrinkled. When the bitch is in full estrus, the vaginal folds have pronounced wrinkles with well defined edges). As the bitch progresses from estrus to diestrus, the vaginal folds become flattened, and the vaginal mucosa takes on a red?and?white?striped appearance. Vaginoscopy is easy to do and can be done in an awake, nontranquilized, standing bitch. Proctoscopes or endoscopes, either flexible or rigid, may be used. The scope must be no more than 8 to 15 mm in diameter and at least 10 to 20 cm in length with an adequate light source. You can use vaginoscopy with or in lieu of vaginal cytology.

Serum progesterone and luteinizing hormone assays

Ovulation timing kits have recently become available for use in veterinary practice. These kits have revolutionized canine breeding management. These kits are invaluable when managing bitches with fertility problems or when limited breedings are required or desired. These kits are easy to use and have a shelf life of about one year when kept refrigerated. They allow the routine use of cooled, shipped semen and frozen semen in bitches. And they've improved the breeding management of bitches with cycles shorter or longer than average.

Ovulation timing kits are enzymelinked immunosorbent assays (ELISA) that detect serum progesterone concentrations. During proestrus, a bitch is under the influence of estrogen but not progesterone. The first rise in progesterone concentration correlates with the ovulatory luteinizing hormone (LH) peak (Figure 2). The combination of estrogen and progesterone initiates standing behavior in bitches. Once the ovulatory LH peak has occurred, ovulation will begin in 24 to 48 hours. It takes about 24 to 48 hours for ovulation to be complete, and it takes another 24 to 48 hours for the primary oocytes to undergo two meiotic divisions to become secondary oocytes capable of being fertilized (Figure 2).

Ovulation timing kits allow indirect detection of the LH peak by detecting the first rise in progesterone concentration. Test bitches every other day, usually starting on Day 5 of proestrus or when the vaginal cytologic examination reveals at least 50% superficial cornified epithelial cells You can start testing earlier if there is any question about the cycle length or if a bitch has a history of a short cycle length. Testing may progress to daily sampling if shipped or frozen semen demands a more accurate detection of the progesterone concentration increase. A tentative plan, once the first increase in progesterone concentration has been detected, is to breed the bitch four and six days later. It is important to keep testing for the continued increase in progesterone concentration. Most bitches will have progesterone concentrations above the upper detection limit of the kit four to six days after the initial increase in progesterone concentration. The upper detection limit of most kits is 5 to 7.5 ng/ml. Occasionally, bitches do not progress as expected. By continuing to test these bitches until their progesterone concentration goes beyond the kit's upper detection limit, breeding can be assured to take place at the appropriate time.

Several ELISA kits are available (e.g. Status Pro Canine Ovulation Timing Test Symbiotics; Target Canine Ovulation Test Biometallics; Ovucheck Camelot Farms). Breeding recommendations for using these kits vary among the type of breeding desired. In our experience, when breeding naturally or with fresh semen by artificial insemination breeding should take place the day that a progesterone concentration of 5 to 7.5 ng/ml is achieved and again 24 to 48 hours later. The same basic recommendation applies to cooled, shipped semen. When using frozen semen, intrauterine deposition of semen is best performed 72 hours after a progesterone concentration of 5 to 7.5 ng/ml is reached. This is the optimal time when the highest numbers of viable secondary oocytes are available for fertilization.

Other uses for progesterone kits make them a worthwhile addition to a practice's laboratory. Progesterone concentration is easy to interpret; any progesterone concentration over 1 ng/ml is indicative of luteal tissue on the ovary. Progesterone assays may provide useful information when pyometra is suspected in a bitch whose cycle history is unknown. In pregnant bitches, a drop in body temperature may signal a drop in progesterone concentration, which indicates the end of the luteal phase and can help you predict the onset of whelping. Progesterone concentration testing can be used to diagnose ovarian remnant syndrome, the presence of ovarian tissue, in a spayed bitch or queen. By measuring progesterone concentrations you can also determine the presence of luteal tissue in a bitch that is cycling silently.

Recently, an LH assay has been marketed (Status LH Luteinizing Hormone Test Synbiotics) for use in breeding management of bitches. The suggested use for this assay is to test blood samples daily starting on Day 5 of proestrus or after vaginal cytologic examination reveals at least 50% cornified epithelial cells. When using the LH assay, remember that a bitch might have numerous LH surges before the ovulatory LH peak. Always use a progesterone assay in conjunction with an LH assay to determine the optimal breeding time. This adds to the cost of the breeding but may be justified in certain situations, particularly when breeding with frozen semen.

Surgical insemination

Surgical insemination in bitches was first introduced when frozen semen was being investigated in dogs. In cows and mares, successes were obtained by intrauterine deposition of thawed semen. Because of the abdominal location of the canine cervix and the perpendicular route of the cervical lumen, it is difficult to access the canine uterus via the vagina. Good conception rates in bitches were obtained only when the frozen semen was deposited directly into the uterus. This is most easily accomplished through a laparotomy incision. We have found that surgical insemination is an acceptable therapy for infertility in bitches. In our experience, a high percentage of bitches that did not successfully conceive after intense breeding management conceived when bred surgically.

The technique for surgical insemination is simple. After routine surgical preparation of the caudal abdomen, make an abdominal incision over the body of the uterus. Exteriorize the body of the uterus through the incision site. Inject semen through the wall of the uterus into the body of the uterus either using a syringe with a 20ga needle or through a 2in, 20ga intravenous catheter placed in a prepunched hole in the uterine wall. You can make the hole by using the blunt end of a suture needle. Fresh, cooled, or frozen semen can be used, although fresh semen is preferred. Inject the semen into the uterine body with a needle or catheter directed toward the uterine horns. You will be able to see and feel the uterine lumen fill with semen. Place digital pressure over the insemination site as you remove the needle or catheter. Then return the uterus to the abdomen, and close the incision site routinely. Timing is critical when using surgical insemination, as with any breeding. Ovulation timing kits are mandatory when using surgical insemination with frozen semen or for treating infertility.

Transcervical insemination

An alternative method of intrauterine insemination is transcervical insemination through the vagina. The canine vagina is relatively long (e.g. 10 to 14 cm in a 24lb [11kg] bitch and up to 29 cm in giant breed bitches). The canine cervix is in the abdomen, and the cervical lumen runs caudoventrally from the uterus to the vagina. Because of these unique anatomical characteristics in dogs, specialized equipment is necessary to catheterize the cervix. Fiberoptic endoscopic equipment for this purpose has to have sufficient length and diameter to access the anterior vagina (Storz extended length fiberscope for urethrocystoscopy Karl Storz Veterinary Endoscopy). With practice, you can learn how to place a catheter in a bitch's cervix so you can deposit semen directly into the uterus. This technique's advantages over surgical insemination include avoiding the stress of anesthesia and surgery and having the ability to do multiple inseminations over several days, which may increase the success of breedings.

Conclusion

Breeding management in dogs has undergone dramatic changes in the last 10 to 15 years. If routine breeding management proves to be insufficient, you can use additional breeding management tools, which now include inhouse progesterone and LH kits. If available, laboratories that provide radioimmunoassay quantitative same day or next day results will provide more accurate information than ELISA kits. With these advances in canine breeding management, the successful use of cooled, shipped semen and frozen semen is becoming routine in dogs.

REFERENCES

1. Nicoletti, P.; Chase, A.: An evaluation of methods to diagnose Brucella canis infection in dogs. Compend. Cont. Ed. 9 00:1071?1073: 1987.

2. Bjurstrom, L.; Linde?Forsberg, C.: Longterm study of aerobic bacteria of the genital tract in breeding bitches. AJVR 53 (5):665669; 1992.

3. Olson, P.N. et al.: The use and misuse of vaginal cultures in diagnosing reproductive diseases in the bitch. Current Tberapy in Theriogenology, 2nd Ed. (D.A. Morrow, ed.). W.B. Saunders, Philadelphia, Pa., 1986; pp 469?475.

4. Bjurstrom, L.; Linde?Forsberg, C.: Longterm study of aerobic bacteria of the genital tract in stud dogs. AJVR 53 (5):670?673; 1992.

5. Strom, B.; Linde?Forsberg, C.: Effects of ampicillin and trimethoprim?sulfamethoxazole on the vaginal bacterial flora of bitches. AJVR 54 (6):891?896; 1993.

6. Olson, P.N. et al.: Concentrations of reproductive hormones in canine serum throughout late anestrus, proestrus and estrus. Biol. Reprod. 27 (5):1196?1206; 1982.

7. Holst, P.A.; Phemister, R.D.: Onset of diestrus in the Beagle bitch: Definition and significance. AJVR 35 (3):401?406; 1974.

8. Lindsay, F.E.F.: 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?15; 1983.

9. Smith, F.O.: Cryopreservation of canine semen. Proc. Soc. 7berio., Society for Theriogenology, Austin, Texas, 1987; pp 249?262.

10. Pineda, M.H. et al.: Dorsal median postcervical fold in the canine vagina. AJVR 34:1487?1491; 1973.

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