Successful pregnancy following gonadotrophin-releasing hormone analogue treatment in a previously infertile bitch.

A Labrador retriever bitch was presented with repeated unsuccessful breeding during both the third and fourth oestruses, even though accurate timing for natural mating was planned based on the results obtained using a semiquantitative luteinising hormone kit and a quantitative assay of progesterone. To identify the problem, serum luteinising hormone, follicle-stimulating hormone, progesterone, and oestradiol-17beta concentrations around the fourth oestrus stage were assayed by radioimmunoassay. Abnormal luteinising hormone fluctuations were detected during pro-oestrus and oestrus, and lower than normal progesterone concentrations were found during the mating period. In the fifth to seventh oestrus, the bitch was successfully treated with gonadotrophin-releasing hormone analogue at times based on the findings of the hormonal assays.

Induction of canine pyometra by inoculation of Escherichia coli into the uterus and its relationship to reproductive features.

To characterize oestrus-related factors affecting the induction of and recovery from pyometra in bitches, 60 clinically healthy beagle bitches were used for induction of pyometra by inoculation of Escherichia coli into the uterus during oestrous and metoestrous stages. The animals were classified into the following six groups according to inoculation time: Days 1-10, 11-20, 21-30, 31-40, 41-50 and 51-60 after LH surge. The incidence of pyometra during the periods Days 11-20 and 21-30 after LH surge was 90.9% and 78.9% respectively, while that during Days 1-10 and 51-60 after LH surge was less than 20%, and the patterns of the incidence of pyometra and the serum progesterone levels were similar. There was no difference in the incidence of pyometra induced in bitches less than 5 years old compared to bitches over 6 years old. Oestrus in all of the bitches with pyometra induced by E. coli returned with or without PGF 2alpha treatment, unlike in bitches with spontaneous pyometra. The duration of the oestrous cycle in the non-treated and PGF 2alpha-treated groups was 231.4+/-55.2 days and 162.1+/-40.6 days (P < 0.001), respectively, and there was no difference in the rate of return of oestrus between the two groups. The conception rate in all of the bitches in which oestrus had returned was 81.8%. The above findings indicate that the period during which severe pyometra could be induced was limited to the early stage in metoestrus.

Three-dimensional reconstruction of the equine ovary.

The equine ovary has a very unique structure in terms of its extreme large size, the presence of the ovulation fossa and the inverted location of its cortex and medulla. In the previous study, it was recognized that the application of three-dimensional internal structure microscopy (3D-ISM) to observe the mare ovary is very effective. Three-dimensional reconstruction of serially sliced images made by 3D-ISM was successful in this study with the aid of the sophisticated image processing technique. The rotation of the reconstructed ovary has been carried out with and without the application of the transparency technique in the ovarian stromal region. The spatial localization of follicles and corpus luteum was clearly visualized by rotating the reconstructed image of the ovary. The extraction of the images of follicles and corpus luteum was also available and gave a quantifiable understanding of their structure.

A case of a bitch with imperforate hymen and hydrocolpos.

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 x 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.

Canine parentage testing based on microsatellite polymorphisms.

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.

Artificial inseminations with frozen semen and application of DNA fingerprint for parents/offspring conformation on Great Pyrenees.

Frozen semen of Great Pyrenees, air-shipped from France, was artificially inseminated (AI) to two Great Pyrenees bitches. Blood LH and P concentrations and vulval width principally determined optimal breeding time of bitches. AI were performed on 17 to 21 days from the onset of estrous bleeding, and 25 days after final Al, pregnancy of one bitch, with two fetuses, was identified by ultrasound image diagnostic equipment. On 62 days, the bitch whelped one male pup and one female pup. Blood samples were obtained from four parents and offspring, and their DNA was extracted. DNA samples digested with four kinds of restriction endonucleases, and resulting fragmented DNA were used as test samples for DNA fingerprinting with Myo prove. As a result bands from pups, displayed by any enzyme, were inherited from either parent, and clear parent-offspring relationships were identified.

Use of microsatellite markers for parentage testing of two litters of beagles born after artifcial insemination with mixed fresh semen.

This study was undertaken to determine if 20 kinds of microsatellite markers, which have already been reported, were useful for the parentage testing of 16 Beagle puppies born after artificial insemination using intentionally mixed semen from six male Beagles. Of the 20 kinds of markers used in this study, 17 kinds (the exceptions were CPH6, FH0020 and FH0419) produced 3 to 5 kinds of alleles, and using these markers, the parentage was finally established for all of the 16 puppies. These 17 kinds of markers showed sufficient amplification in PCR and had no non-specific PCR products or alleles with single-base-alterations, which makes them very useful as markers for identification of individual Beagles and useful for parentage testing.

Shrinkage in the horizontal dimensions of the vulva (vulvar shrinkage) as an indicator of standing heat in the beagle.

During the proestrous and estrous periods in 12 beagles, the following parameters were measured daily: the horizontal dimensions of the vulva, vaginal cytology, and serum luteinizing hormone (LH) and progesterone concentrations. Measurements of serum LH concentrations allowed for the identification of the LH surge and the optimal time for artificial insemination (AI). Nine out of the 12 beagles became pregnant through AI and completed a gestation. Shrinkage in the horizontal dimensions of the vulva (i.e., vulvar shrinkage) was primarily observed prior and subsequent to the LH surge. In six of the nine (66.7%) beagles that became pregnant, vulvar shrinkage was observed on the days in which the LH surge was confirmed, and the rate of vulvar shrinkage tended to be greater at higher serum LH concentrations. Further vulvar shrinkage was identified in all nine beagles within two days of the LH surge. An increase in the serum progesterone concentration was observed after the LH surge in each of the beagles that became pregnant, together with clinical signs of estrous behavior (i.e., standing heat) as well as a change in vulva condition from swollen to soft. This demonstrates that vulvar shrinkage is induced in response to the onset of the LH surge and that the LH surge can be predicted through the measurement of the horizontal dimensions of the vulva, vaginal cytology, and the assessment of serum progesterone concentrations in beagles.

Determination of optimal time for mating by artificial insemination with chilled semen using luteinizing hormone surge as an indicator in beagles.

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).

Immunohistochemical study of steroidogenic enzymes in the ovary and placenta during pregnancy in the dog.

Using the immunohistochemical technique, we attempted to identify the source of secretion of steroid hormones between the mid- and late-terms of gestation in dogs by investigating steroid converting enzymes such as cholesterol side-chain cleavage enzyme (SCC), 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), 17 alpha-hydroxylase/C17, 20lyase (c17), and aromatase in the ovaries and placenta. Aromatase positive cells were slightly confirmed in luteal cells in the mid-term of gestation (day 40), whereas, in the late-stage (day 50 and 60), the number of aromatase positive cells had increased. However, the oestrogen precursor (c-17 positive cells), could barely be identified in the marginal regions of the corpora lutea (CL) and completely disappeared in the late-stage of gestation. The androgen precursors, convertase SCC and 3 beta-HSD, were confirmed in all regions of the CL during the mid-stage of gestation (day 40), showing particularly strong cell reactions in the marginal region of the CL. Yet, these positive reactions of SCC and 3 beta-HSD in the marginal region of the CL disappeared in the late-stage of gestation. Moreover, it was discovered that the number of SCC and 3 beta-HSD positive cells had decreased in all regions of the CL. None of the enzymes were detected in the placenta. The above results indicated that the source of oestrogen secretion in pregnant dogs is considered to be the CL, and that, compared with the mid-stage of gestation, there was an increased number of oestrogen synthesizing cells within the CL in the late-stage. However, the biosynthetic site of oestrogen precursors from the luteal cells during the late-stage of gestation is still unknown.

3 beta-Hydroxysteroid dehydrogenase activity and gestagen concentrations in bovine cotyledons and caruncles during gestation and parturition.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) activity in bovine cotyledons was much higher than in caruncles throughout the gestation period. The activity of this enzyme in cotyledons increased greatly to 150.6 +/- 5.8 pmol min-1 mg-1 protein during the seventh month of gestation, reached a peak of 221.0 +/- 34.9 pmol min-1 mg-1 protein during the eighth month, and decreased at parturition. Progesterone and 20 alpha-hydroxyprogesterone concentrations in cotyledons also increased sharply to 2.69 +/- 0.30 and 2.15 +/- 0.42 ng mg-1 protein, respectively, during the seventh month of gestation, reaching peaks of 2.86 +/- 0.47 and 2.51 +/- 0.36 ng mg-1 protein, respectively, during the eighth month and decreasing at parturition, in a manner similar to the activity of 3 beta-hydroxysteroid dehydrogenase. The fluctuation of 17 alpha-hydroxyprogesterone concentration in cotyledons was different from that of progesterone and 20 alpha-hydroxyprogesterone. These findings indicate that the activity of 3 beta-hydroxysteroid dehydrogenase in the placenta is enhanced during the third trimester, and progesterone synthesized in the cotyledons is converted concurrently to 20 alpha-hydroxyprogesterone before progesterone is transferred to the fetal blood.

Aromatase activity and oestrogen concentrations in bovine cotyledons and caruncles during gestation and parturition.

Two peaks of aromatase activities were detected in bovine cotyledons, 2.76 +/- 0.96 pmol min-1 mg-1 protein at month five of gestation and 3.62 +/- 0.78 pmol min-1 mg-1 protein immediately after parturition, but aromatase activity in caruncles was very low throughout gestation. Oestrone concentrations in the cotyledons and caruncles were 0.93 +/- 0.50 and 1.51 +/- 0.69 ng mg-1 protein at month five of gestation, but 4.31 +/- 1.66 and 3.36 +/- 0.98 ng mg-1 protein immediately after parturition, indicating a biphasic pattern. Our findings suggest that oestrogen synthesis in the bovine placenta increased to a maximum at least twice during the period from gestation to parturition; at month five of gestation and at parturition the changes in aromatase activity may only be partially explained by changes in oestrogen production.

Changes of plasma concentrations of steroid hormones, prostaglandin F2 alpha-metabolite and pregnant mare serum gonadotropin during pregnancy in thoroughbred mares.

Plasma concentrations of estrogens, gestagens, cortisol (F), 13, 14-dihydro, 15-keto PGF2 alpha (PGFM) and pregnant mare serum gonadotropin (PMSG) in 10 Thoroughbred mares were measured for a 11-month pregnancy period. Estrone (E1) and estradiol-17 beta (E2) levels gradually increased as the pregnancy advanced, showing a peak around Month 8 and decreased thereafter. Progesterone (P) levels increased on Months 3 and 11, and 17 alpha-OH-progesterone (17 alpha-OHP) levels peaked on Month 3, whereas 20 alpha-OH-progesterone (20 alpha-OHP) levels increased sharply after Month 6. PGFM indicated peaks on Months 2 and 11. F and PMSG levels peaked on Months 2 and 3. From factor analysis, Month, E1, E2 and 20 alpha-OHP were discriminated as Factor 1, increasing with the progress of pregnancy, PMSG, 17 alpha-OHP and P as Factor 2, showing a relation with the secondary corpus luteum, and PGFM and F as Factor 3, affecting PGFM change on Month 2 by F. P also related to both Factors 1 and 3, showing an inverse relation against PGFM. In conclusion E1, E2 and 20 alpha-OHP contained in Factor 1 were suggested to be important especially as parameters of placental function after Month 6.

Adrenocorticotropin and cortisol levels in the plasma of bovine fetuses and neonates.

Plasma ACTH and cortisol levels in the bovine fetuses over the period of 5 to 9 months of gestation and in the neonates immediately after birth and at 5 days old were studied. In the bovine fetuses, the plasma ACTH levels ranged from 60.8 +/- 17.8 to 71.3 +/- 19.7 pg/ml over the period of 5 to 7 months of gestation. It increased rapidly to 239.2 +/- 261.5 pg/ml at 8 months and to 406.9 +/- 409.4 pg/ml at 9 months of gestation. In the neonates immediately after birth it decreased to 182.3 +/- 110.7 pg/ml. The plasma cortisol levels ranged from 3.23 +/- 2.12 to 3.85 +/- 2.52 ng/ml over the period of 5 to 8 months of gestation and increased to 8.10 +/- 4.88 ng/ml at 9 months of gestation. It then increased rapidly to 88.35 +/- 42.78 ng/ml in the neonates immediately after birth. The correlation between plasma ACTH and cortisol levels in the fetuses of 5 to 7 months of gestation was not significant, but in the fetuses of 8 and 9 months of gestation and neonates were significant. However, especially immediately after birth, the increase in plasma cortisol occurred without a concomitant rise in plasma ACTH. According to these findings, it is suggested that the pituitary-adrenocortical axis in bovine fetus matures in the later stage of gestation and an increase of sensitivity in the fetal adrenal gland to ACTH may serve as a trigger for the onset of parturition.