ABSTRACT. To establish an accurate method for parentage testing in dogs, microsatellite DNA repeat length polymorphisms were examined. We selected twenty microsatellite markers reported previously and examined their application for parentage testing in Beagles and Labrador Retrievers. Heterozygosity (He), Polymorphism Information Content (PIC), the probabilities of Paternity Exclusion (PE) and the combined PE were calculated from allelic frequencies of the markers. All markers amplified by polymerase chain reactions were polymorphic and many markers showed high He and PIC in the both breeds. The final combined PEs in Beagles and Labrador Retrievers were 0.999994 and 0.999920, respectively. The results suggest that the twenty markers can be applied for routine parentage testing in dogs.

ABSTRACT. A 38-month-old female Golden retriever was presented with dysuria and dyschezia. It was difficult to visualize the vagina by vaginoscopy due to a cystic polyp on the hymen. The polyp was 2 × 3 cm in diameter, round, and pink in color. From clinical and imaging evaluations the original diagnosis was mucometra or pyometra. From endoscopic examination of the vagina an imperforate hymen was finally diagnosed. The ovaries, uterus, and half of the vagina were removed through a median abdominal incision. The vagina contained about 1.5 liters of fluid, but the uterus and ovaries appeared normal. This is a rare case with imperforate hymen and hydrocolpos with a polyp on the hymenal membrane in bitch. KEY WORDS: bitch, hydrocolpos, imperforate hymen.J. Vet. Med. Sci. 63(4): 475-477, 2001 Congenital abnormalities of the canine vagina and vulva are classified into six categories, including four vestibulovaginal ring strictures and two strictures in the vestibulovulvar area [12]. The female genital tract is formed from the Mullerian ducts and urogenital sinus [9]. The hymen is formed by fusion of the Mullerian ducts and urogenital sinus, and in domestic animals disappears during the fetal period or after birth [4,5,9]. An imperforate hymen causes hydrocolpos, hydrometra and hydorosalpinx (also hemato-, respectively) [10]. A similar accumulation of vaginal fluid is also caused from vaginal atresia and vaginal aplasia [10]. In the bitch and queen, there are a few reports of imperforate hymen [7,11] and vaginal atresia [2,7,13]. We report a Golden retriever bitch with imperforate hymen and hydrocolpos due to a polyp on the hymen, diagnosed by endoscopic examination.A 38-month-old female Golden retriever weighing 30 kg was presented to the Animal Medical Center, Nihon University, with dysuria and dyschezia. The dog had been treated successfully for vaginitis and cystic calculus at 3 months and 2 years of age, respectively. During estrus, 3 months prior to presentation, the owner noticed vulvar swelling (a normal estrous sign), but without proestrous bleeding.Physical examination revealed a normal bitch except for the cystic polyp in the vagina. Urinalysis revealed a pH 6, slight protein and occult blood, 20/hpf red blood cells and 8/ hpf white blood cells consisting mainly of neutrophils were observed in the urine sediment, and the urine specific gravity was 1.030. The size of the cystic polyp prevented full digital exploration of the cranial vagina, but no vulvar discharge was noted. Left lateral (Fig. 1-A) and ventrodorsal ( Fig. 1-B) radiographs of the abdomen revealed an extremely enlarged vagina (15 × 27 cm), initially diagnosed as the uterus, located above the urinary bladder and reaching to the posterior margin of the liver. On the ventrodorsal view, the cranial pole of the urinary bladder extended to the last rib, displaced toward the right side of the abdomen, with a very long proximal urethra. The vagina, which was thought to be a uterus simplex, displaced to the left side, occupied two-thirds of the abdom...

ABSTRACT. Parentage testing was performed in sixteen litters by canine artificial inseminations with frozen semen from different sires on Days 5 and 7 after the LH surge. It became apparent that only 25% of dams had superfecundation, but 43.8% of dams were whelped after insemination only on Day 5 after the LH surge and 31.3% of dams after insemination only on Day 7. Of the total 87 puppies , 46% were born after insemination on Day 5 after the LH surge and 54% after insemination on Day 7. This result strongly suggested that canine artificial insemination with frozen semen could be sufficiently successful also on Days 5 and 7 after the LH surge. KEY WORDS: canine, optimal time, parentage testing.J. Vet. Med. Sci. 65(9): 1003-1005, 2003 Canine ovulation occurs 48 hr after the LH surge in most bitches [2,13,19]. Ova are fertilizable from 60 hr (2.5 day) to 108 hr (4.5 day) after ovulation [18]. The estimated intrauterine fertile lifespan of frozen-thawed semen is <24 hr [1] and the sperm require 7 hr for capacitation [11]. Therefore, theoretically the optimal time for insemination with frozen semen would be around Day 4 after the LH surge [9] and Days 4 and 7 after the LH surge have previously been recommended [10]. Nishiyama et al. [12] reported that the conception rate from inseminations on Days 5 and 7 after the LH surge was at least twice as high as that from inseminations on Days 4 and 6. This study investigated which day had the higher conception rate when insemination with frozen semen was performed twice, on Days 5 and 7, after the LH surge. To discriminate the individual sire on Days 5 and 7 after the LH surge, frozen semen with a fertile lifespan within 24 hr was used and parentage testing by microsatellite markers was performed.Twenty female and 8 male beagles keeping at the Laboratory of Theriogenology of Nihon University were used for this study. The ages of the bitches and sires were 3.7 ± 1.2 (1.2-5.5) and 3.4 ± 1.4 (1.8-5.8) years, respectively. The animals were maintained in an animal room and housed in individual cages. They were supplied dry food (Science Diet TR , Maintenance, Hill's and Corgate Japan Co., Tokyo, Japan) once a day and drinking water ad libitum.Only the second fraction of ejaculate was collected by manual manipulation for use as frozen semen. The volume, concentration, color, progressive motility and abnormality were estimated in undiluted semen. The semen was centrifuged (500 g, 5 min) and the sperm-rich fraction was extended with modified TRIS-yolk-citrate extender, twice. The number of washed sperm was adjusted to 2 × 10 8 /ml with modified TRIS-yolk-citrate extender at 30°C and cooled to 4°C stepwise at -0.3°C/min in an automatic freezer. The solution was added to the same total volume of 6% glycerol-TRIS-yolk-citrate extender with an additional one-fourth at 10 min intervals, placed in 0.5 ml plastic straws and kept for 90 min for equilibration. The straws were placed horizontally 5 cm above the surface of liquid nitrogen fill in a styrofoam box, for 6 min and subsequently pl...

ABSTRACT. CD56+ cells in canine blood leukocytes were characterized by flow-cytometric analysis of peripheral blood of 30 healthy adult beagle-dogs (15 males and 15 non-pregnant females). In 19 of the 30 dogs, anti human CD56 antibody, reacted CD56, which is a neural cell adhesion molecule (N-CAM), is a membrane glycoprotein belonging to the immunoglobulin superfamily [3,8]. CD56 is expressed mainly on natural killer (NK) cell in human peripheral blood and is used as a surface marker of human NK cell [2]. Although the precise functional role of CD56 molecules on human NK cells is not fully understood, CD56 is thought to be involved in cell-attachment of NK cells to the target cells in their cytokilling process [10].In rhesus monkeys, however, peripheral blood NK cells predominantly show CD56 -phenotype [1]. Furthermore, CD56 is expressed on almost all peripheral blood monocytes in cynomolgus monkeys [9,12] and in rhesus monkeys [1]. Therefore, CD56 is not always a specific marker for NK cells in mammals other than humans.An anti human CD56 antibody, Leu-19 (Becton Dickinson, San Jose, California, U.S.A.), cross-reacts with canine leukocytes [14]. However, detailed characteristics of canine CD56 + cells remain unclear. In this study, we clarified the phenotypic characteristics of canine CD56 + cells by flow-cytometric analysis on canine peripheral blood leukocytes.Thirty clinically healthy beagle-dogs (15 males and 15 non-pregnant females) 1.4 to 7.3 years old were used in this study. By vaccination, all dogs were free from rabies virus, canine distemper virus, canine herpesvirus, canine parvovirus, canine parainfluenza virus, and canine adenovirus. Blood was collected from each dog's jugular vein, using sodium citrate as an anti-coagulant. We confirmed that the complete blood counts and the leukocyte differential counts of all blood samples were within normal ranges (data not shown).Direct staining of peripheral blood leukocytes was done as follows. The following pairs of antibodies were reacted with 50 µl samples of blood for 15 min at room temperature: anti human CD56 phycoerythrin conjugated (-PE) antibody (Leu-19; Becton-Dickinson) and anti canine CD3 fluorescein isocyanate conjugated (-FITC) antibody (CA17.2A12; Serotec, Oxford), anti human CD21-PE antibody (B-ly4; Becton-Dickinson) and anti canine CD3-FITC antibody, or isotypic control-FITC antibody (Beckman-Coulter, Fullerton, California, U.S.A.) and isotypic control-PE antibody (Beckman-Coulter). To each of these samples, we added 1.5 ml of Erythrocytes Lysing Buffer (0.83% ammonium chloride, 0.1% potassium hydrocarbonate, and 0.004% EDTA disodium salt in distilled water), and then left the sample for 7 min at room temperature. After centrifugation, the supernatant was removed by aspiration. The pellet was washed twice with wash buffer (phosphate buffered saline containing 0.5% bovine serum albumin, 2 mM EDTA disodium salt, and 0.03% sodium azide) at 4°C. The samples were fixed with 1% paraformaldehyde-phosphate buffered saline, and then subjected to ...

Artificial insemination (AI) was conducted using the second fraction of semen, which was collected from 15 male dogs, diluted to a total sperm count of 100x10(6) for each insemination with egg-yolk Tris (eyT) citrate acid buffer and incubated at 4 degrees C for 48 hours. Luteinizing hormone (LH) surge was detected to determine the optimal time for mating using canine LH assay kits. Artificial insemination using 100x10(6) sperm was performed on the fourth and sixth days or the fifth and seventh days after the LH surge. The conception rates were 33% (4/12) and 89% (8/9), respectively; the whelping rates also showed similar results. Serum LH and follicle stimulating hormone (FSH) concentrations were measured in nine dogs, and the mean LH concentration (+/- standard deviation) at LH surge was 15.77+/-7.66 ng/ml. The time of the LH surge detected by the canine LH assay kit was very similar to that measured by radioimmunoassay (RIA).