Presence of surfactant proteins in the uteri and placentae of pregnant mares.

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.

Changes in luteinizing hormone pulse frequency and prolactin levels in bitches in response to estrus induction by cabergoline-its cases where it is delayed to induce estrus.

The effect of estrus induction by cabergoline on gonadotropin and steroid hormone responses was examined in anestrous bitches. Eleven beagles were used in the study; seven were included in the estrus induction group and four were included in the spontaneous estrus group. Cabergoline was orally administered to the estrus induction group at 5 µg/kg once daily for four weeks, or until hemorrhagic discharge was detected. The inter-estrus interval in the estrus induction group was significantly shorter than the previous estrus interval. Bitches that showed proestrus within four weeks of treatment showed increased luteinizing hormone (LH) pulse frequency and, subsequently, increased estradiol (E2) levels. Prolactin (PRL) levels declined promptly after treatment, except in one bitch that did not show proestrus during the cabergoline treatment period. There was a significant correlation between the time to proestrus induction and the reduction in PRL levels. A positive correlation was found between the LH levels two weeks after cabergoline administration and PRL reduction. This study demonstrates that an abrupt reduction in PRL is likely to be important for initiation of estrus in bitches. A reduction in PRL indirectly leads to an increase in LH pulse frequency, which regulates follicular development in bitches. However, if the period from the end of the previous estrus to the cabergoline treatment is short, it may take some time to show proestrus without increasing E2 levels, even if the LH level increases after cabergoline administration.

Serum concentrations and testicular expressions of insulin-like peptide 3 and Anti-Müllerian hormone in normal and cryptorchid male horses.

Insulin-like peptide 3 (INSL3) is an important hormone for testicular descent during embryonic development and a factor for assessing functional status of Leydig cells of testes, but there is limited number of equine studies. Anti-Müllerian hormone (AMH) is a useful diagnostic marker for cryptorchidism in horses. This study aimed to compare serum concentrations and testicular expression intensity of INSL3 and AMH in intact and cryptorchid male horses. Serum INSL3 concentrations in intact (n = 9; mean ± SEM, 19.9 ± 5.9 ng/mL) and noncastrated unilateral cryptorchid (UC) male horses (n = 16; mean ± SEM, 16.8 ± 4.1 ng/mL) were higher compared with hemicastrated unilateral cryptorchid (HCUC) male horses (n = 9; mean ± SEM, 3.8 ± 0.7 ng/mL) (P < 0.05). And serum INSL3 in bilateral cryptorchid (BC) male horses (n = 4; 1.9 ± 0.4; mean ± SEM, ng/mL) were lower compared with intact male horses (P < 0.05). Serum AMH concentrations in BC male horses (n = 3; mean ± SEM, 30.6 ± 4.8 ng/mL) were higher compared with intact male horses (n = 5; mean ± SEM, 12.2 ± 3.9 ng/mL) (P < 0.05). Immunostaining of scrotal and cryptorchid testis showed that Sertoli cells were positive for AMH, and Leydig cells were positive for INSL3. Staining intensity of AMH was higher in cryptorchid testis than in scrotal testis (P < 0.05). Furthermore, AMH expression intensity was higher in abdominal testis than in inguinal testis (P < 0.05). Immunostaining intensity of INSL3 in the testis was positively correlated with serum INSL3 (r, 0.7; P < 0.01), seminiferous tubule area (r, 0.727; P < 0.01), and Johnsen score for spermatogenesis (r, 0.604; P < 0.05), whereas immunostaining intensity of AMH in the testis was negatively correlated with seminiferous tubule area (r, -0.814; P < 0.01) and Johnsen score for spermatogenesis (r, -0.807; P < 0.01). Our findings suggested that AMH is a good biomarker for diagnosing cryptorchidism in male horses, in addition to INSL3 values to assess the testis of intact and cryptorchid male horses.

Effect of high-concentrate corn grain diet-induced elevated ruminal lipopolysaccharide levels on dairy cow liver function.

A high-concentrate diet destroys gram-negative bacteria in the cattle rumen, leading to elevated ruminal lipopolysaccharide (LPS) levels. LPS causes liver inflammation through the hepatic portal vein but little is known about the effects of rumen-derived LPS on liver function and the reproductive organs. In this study, we determined the effect of increasing rumen fluid LPS levels on liver function and genital LPS levels. Cows were assigned to control (CON; n=5) and high-concentrate diet (HC; n=7) groups. We observed that the ruminal LPS and haptoglobin (Hp) levels were significantly higher and albumin levels were lower in the HC group than in the CON group. In the HC group, The Hp levels and aspartate transaminase (AST) activity were significantly higher and the total cholesterol levels were significantly lower after high-concentrate diet feeding than before feeding. No differences were observed in LPS levels in the peripheral veins, hepatic veins, hepatic portal vein, uterine perfusate, and follicular fluids between the groups. In all samples, the LPS level in the hepatic portal vein blood positively correlated with the AST activity and serum amyloid A level. In conclusion, our results indicate that high-concentrate diets do not have a direct effect on the reproductive organs upon a moderate ruminal LPS level increase. However, an increased ruminal LPS influx into the liver might affect negatively liver function.

Effect of feed restriction on hepatic estradiol metabolism and liver function in cows.

In this study, the effects of restriction feeding on the liver function, hepatic uridine diphosphate glucuronosyltransferase (UGT) activity, hepatic insulin-like growth factor (IGF)-1 mRNA expression and response to high-dose estradiol-17ß (E2) administration were investigated in non-lactating cows. Cows were assigned to either restricted feeding (30% of total digestible nutrient requirement) or ad libitum feeding of a dent corn-based concentrate and roughage for a 2-week feeding trial (Day 1=day of beginning the feeding trial). On day 14, a high-dose E2 administration study was carried out to examine plasma E2 levels as an indicator of hepatic E2 metabolism. Plasma E2 concentration in the restricted feeding group was consistently higher after high-dose E2 administration than in the control group. In addition, indocyanine green half-life value was prolonged by restricted feeding for 13 days, and increased liver triglyceride concentration and decreased liver UGT activity were caused by this restriction over 14 days. Restricted feeding did not affect plasma IGF-1 concentration or hepatic IGF-1 mRNA expression. These results suggest that two weeks of restriction feeding led to accumulation of triglyceride, decreased liver blood flow, and slightly impaired liver function, which in turn slowed down the hepatic metabolism of E2 without significantly impacting hepatic IGF-1 production.

Association between liver failure and hepatic UDP-glucuronosyltransferase activity in dairy cows with follicular cysts.

Uridine 5'-diphospho-glucuronosyltransferase (UGT) liver activity was measured using estradiol-17ß as a substrate in dairy cows with follicular cysts. The activity was significantly lower than that in dairy cows with normal estrous cycles (P<0.01). Liver disorders, such as fatty liver and hepatitis, were observed in half cows with follicular cysts, and liver UGT activity was lower than that in cows with normal estrus cycles. In addition, the liver UGT activity was significantly lower in dairy cows with follicular cysts without liver disorders than in dairy cows with normal estrous cycles. Therefore, the cows were divided into those with low, middle and high liver UGT activities, and liver disorder complication rates were investigated. The complication rate was significantly higher in the low- (78.1%) than in the middle- (22.2%) and high-level (8.3%) groups, suggesting that liver disorders are closely associated with the development of follicular cysts in dairy cows and that steroid hormone metabolism is delayed because of reduced liver UGT activity, resulting in follicular cyst formation. We conclude that reduced estradiol-17ß glucuronidation in the liver and liver disorders are associated with follicular cyst occurrence in dairy cows.

Development of ovarian diseases in dairy cows with a history of fatty liver, and their prognosis.

In the present report, the incidence and prognosis of ovarian diseases were evaluated in correlation to the grades of the fatty livers (FL). 233 animals were diagnosed with a FL, based on fat deposition of >10% into the hepatic parenchyma obtained from liver biopsy, and were eventually cured of the FL. These 233 animals were classified into mild group (n=99: hepatic fat deposition rates ranging from 10 to <30%), moderate group (n=56: hepatic fat deposition rates ranging from 30 to <60%) and severe group (n=78: hepatic fat deposition rates of >60%). The incidence of ovarian diseases and the culling rate were, respectively, 40.0% and 10.0% in mild group, 58.3% and 37.5% in moderate group, and 84.9% and 69.7% in severe group. The incidence of ovarian diseases and the culling rates in severe group were significantly (P<0.05) higher than those in mild and moderate groups. It was, therefore, concluded that the elevated incidence of ovarian disease and culling rates tend to be dependent on the higher severity scores of the fat deposition rates in dairy cows with FL and that cases with severe FL, with a fat deposition rate of 60% or higher, were greatly associated with death and culling.

Analysis of the equine ovarian structure during the first twelve months of life by three-dimensional internal structure microscopy.

A three-dimensional internal structure microscopy (3D-ISM) can clarify the anatomical arrangement of internal structures of equine ovaries. In this study, morphological changes of the equine ovary over the first 12 months of life were investigated by 3D-ISM in 59 fillies and by histological analysis in 2 fillies. The weight and volume of the paired ovaries initially decreased from 0 to 1 months to 2 to 3 months of age and then significantly increased at 8 to 12 months of age. The ovulation fossa was first observed around the 3rd month and became evident after the 6th month. The number of follicles with a diameter of ≥10 mm and the diameter of the largest follicle increased gradually after 6 months of age. On a volume basis, the medulla accounted for nearly 90% of the whole ovary at 0 to 1 months of age, but significantly decreased from 2 to 3 months of age. The volume of the cortex increased progressively after birth and reached approximately 60% of the total volume at 8 to 12 months of age. This significant development of the cortex coincided with the increased number and size of large follicles observed from 6 months of age. These results suggest that the development of the cortex plays a role in the maturation of the follicles and the equine ovary undergoes substantial morphological changes postnatally until puberty.

GnRH analogue treatment on LH surge day 0 followed by single transvaginal artificial insemination with frozen semen on day 5 in bitches.

Reproductive parameters were evaluated in 19 and 14 estrous beagles that received 100 µg of gonadotropin-releasing hormone (GnRH) and saline treatment, respectively, on the day of luteinizing hormone (LH) surge (Day 0; estimated by serial progesterone assay) and balloon catheter-aided single transvaginal artificial insemination of frozen semen on Day 5. Although the conception rate and litter size were similar between the GnRH and saline groups, the concentration of LH peak was significantly higher in GnRH-treated bitches (P<0.01). In addition, the actual LH surge did not occur on the estimated Day 0 in one saline-treated bitch. In clinical practice that daily progesterone assay is difficult, administration of GnRH on estimated Day 0 would be recommended to induce or enhance the LH surge for timely and successful insemination.

Evaluation of transcervical insemination using frozen semen by flexible endoscope in dogs.

We evaluated transcervical insemination (TCI) using frozen semen by flexible endoscopy in dogs. Eight female and eight male beagles were used in this study. A flexible endoscope and a washing tube were used for TCI. A tracheal tube was used as an alternative to the penis and was an auxiliary for inserting the flexible endoscope. The mean time required to insert the washing tube into the external os of the uterus after inserting the endoscope into the tracheal tube was 7.5 min. Slight or mild pain was observed in all bitches during TCI. However, TCI could be easily performed with retention in all bitches and without sedation anesthesia. The tracheal tube was useful to ensure the visual field using air sufflation. Clinical signs suspicious of infection were not observed in any bitches from the TCI to the pregnancy diagnosis. The conception rate was 87.5%, and the mean number of fetuses was 6.3. TCI using a flexible endoscope in bitches was performed quickly with minimal invasiveness. We present a new method of TCI in dogs. This method should be studied in small and large breeds to obtain more detailed results.

Relationship between indocyanine green clearance test and feed intake and impaired hepatic function in dairy cows.

The purposes of this study were 1) to examine changes in the indocyanine green (ICG) clearance by feeding and 4-day fasting in dry cows, and (2) to investigate the relationship between ICG clearance and blood chemistry profile in postpartum cows. In 3 dry cows, post-feeding ICG half-life (T(1/2)) was shorter than the pre-feeding value, and post-fasting T(1/2) was longer than pre-feeding and post-feeding values. In 16 lactating cows, T(1/2) value at 2 weeks postpartum showed positive correlations with AST, T-Bil and γ-GTP. These results suggested that ICG clearance correlated with T-Bil and liver enzymes can be sensitive and accurate diagnostic marker for impaired liver function in dairy cows. In addition, ICG clearance is greatly affected by feeding and fasting.

Effect of short-term fasting on hepatic steroid hormone metabolism in cows.

In the present study, the effect of 4-day fasting on steroid hormone metabolism in the liver and secretion of LH was examined in cows. Six non pregnant, dry Holstein cows were used. The estrous cycle was synchronized in all cows using CIDR-Ovsynch. Cows were allocated to a control group (n=3) and a fasting group (n=3). In the fasting group, cows were fasted for four days from day -4 to day -1 (day 0=day of 2nd GnRH injection) but otherwise were fed ad libitum. The experiment was repeated in a crossover design after an interval of about one month. The peripheral progesterone (P(4)) concentration in the fasting group was significantly higher than in the control group on day -1 and 0. The peripheral estradiol-17ß concentration in the fasting group was also significantly higher than in the control group on day -1 and 0. The portal vein P(4) concentration in the fasting group was significantly higher than in the control group. On day 0, there was no difference in LH secretion between groups. The mean percentages of lipid droplets in liver cells in the fasting group were significantly higher than in the control group on day 0. These results suggest that short-term fasting leads to reduced hepatic steroid hormone metabolism by accumulation of fat in the liver, which causes high peripheral steroid hormone concentrations.

Flow cytometric analysis of canine umbilical cord blood lymphocytes.

Lymphocyte subsets in canine umbilical cord blood were flow cytometrically analyzed and compared with those of the dams' peripheral blood. The proportion of CD3+ T lymphocytes, CD21+CD3- B lymphocytes, and CD3-CD21- non-T non-B lymphocytes in umbilical cord blood was 52.9%, 30.4%, and 16.7%, respectively. T lymphocyte/B lymphocyte ratio was significantly lower in the umbilical cord blood than in the dams' peripheral blood (2.1 +/- 1.4 versus 11.0 +/- 8.1, P < 0.001). In contrast, CD4+ lymphocyte/CD8+ lymphocyte ratio was significantly higher in the umbilical cord blood than in the dams' peripheral blood (7.6 +/- 2.2 versus 1.8 +/- 0.6, P<0.001). These findings clarified the phenotypic characters of canine umbilical cord blood lymphocytes.

Characterization of CD34+ cells from canine umbilical cord blood, bone marrow leukocytes, and peripheral blood by flow cytometric analysis.

Characterization of CD34+ cells in canine bone marrow, umbilical cord blood, and peripheral blood was performed by flow cytometric analysis. The ratio of CD34+CD45hi cells, which are absent in human blood, was high in the CD34+ cell fraction, but 98% of these was suggested B-cells. The remaining CD34+CD45lo cells may comprise canine hematopoietic progenitor cells, and these cells accounted for 0.23 +/- 0.07% of the fraction in cord blood, 0.30 +/- 0.07% in bone marrow, and 0.02 +/- 0.01% in peripheral blood.

Clinical significance of circulating vascular endothelial growth factor in dogs with mammary gland tumors.

Increase in circulating vascular endothelial growth factor (VEGF) is suggested as a prognostic indicator in human patients with malignant tumors. The purpose of this study was to evaluate the clinical significance of circulating VEGF in dogs with mammary gland tumors (MGT). Both plasma and serum VEGF were significantly higher in dogs with MGT when compared with those in the healthy dogs. In dogs with MGT, the plasma and serum VEGF of the malignant group increased significantly compared with those of the benign group. Additionally, there was a significant difference between the plasma and serum VEGF in the groups with postoperative metastasis and no metastasis. Circulating VEGF is expected to be clinically available for the determination of prognosis in canine MGT.

Optimum dose of LH-RH analogue Fertirelin Acetate for the induction of superovulation in mice.

The optimum dose for establishing superovulation in mice of Fertirelin Acetate (FA), an LH-RH analogue, was examined. Mice were subcutaneously injected with 5 IU of hCG at 17:00 (Day 0), and with various doses of FA (0.001 to 1.0 microg) five times at 4 h intervals on and after 22:00 on Day 0. To induce ovulation, 5 IU of hCG was again injected subcutaneously at 17:00 on Day 2. In the groups administered with doses ranging from 0.01 to 0.5 microg of FA, the number of ovulated eggs was significantly (p<0.05) larger than in the control group (12.9 +/- 5.9). The greatest number of ovulated eggs (22.6 +/- 7.3) was obtained in the group administered with 0.025 microg of FA. The results indicate that the effective dose of LH-RH analogue, FA, is between 0.1 and 0.5 microg for superovulation induction in mice.

Ovarian teratoma with a formed lens and nonsuppurative inflammation in an old dog.

A 9-year, 7-month-old female German shepherd weighing 26.6 kg was admitted to the hospital for pica and diarrhea. A large mass was found in the right ovary and removed, and cross section of the mass revealed a multilobular tumor consisting of several cystic cavities which contained tufts of dark hair in thick creamy-white sebaceous fluid. Histologically, the tumor consisted of adipose tissue, central nervous tissue, crystalline lens, cartilage and bone. In the central nervous tissue, lens and lesions like nonsuppurative inflammation comprizing of accumulation of glial cells and lymphocytic perivascular cuffing were observed. The tumor was diagnosed as a mature cystic teratoma.

Decreased apoptotic polymorphonuclear leukocyte rate in dogs with pyometra.

Polymorphonuclear neutrophil (PMN) apoptosis was examined in three dogs with pyometra by TUNEL assay in a 24-hr incubation period and compared with that in healthy control dogs (n=5). The incidence of apoptotic PMNs in dogs with pyometra was 26.4 +/- 5% and that in healthy dogs was 54.3 +/- 7%. The results indicated that apoptotic PMN rates in dogs with pyometra were significantly lower than those in control dogs (p<0.05), suggesting the prolongation of PMN survival.

Optimal timing for canine artificial insemination with frozen semen and parentage testing by microsatellite markers in superfecundency.

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.

Development of a method of diagnosing ovarian disorders in sows and gilts using uterine ultrasonography.

In ultrasonographic diagnosis of ovarian disorders and the estrous cycle in sows, transverse observation of the uterus yielded more characteristic findings than observation of sagittal sections. Transverse ultrasonography revealed that the low progesterone (P) type uterus showed a round structure, while the high P type uterus showed a flattened structure. These results corresponded well with rectal palpation findings: the low P type uterus had a hard, pipe-like structure and the high P type a soft, balloon-like structure. For gilts, we employed a minimum convex type transrectal prostate probe that had an approximately 18 cm insertion handle. The images of the uterus obtained thereby were a similar to those obtained from sows. The above results suggest that it should be possible to diagnose and treat many ovarian disorders in sows and gilts based only on the ultrasonograhic findings. In short, ultrasonograhic findings of a round structure of the uterine wall might be an indication for PMSG (pregnant mare serum gonadotropin) treatment, while findings of a flattened structure might be an indication for PGF(2) (alpha) administration.