Circulating testosterone and inhibin levels at different ages in the male beluga (Delphinapterus leucas)

This study is the first report on circulating testosterone and inhibin levels in a species of whales, the beluga. Circulating testosterone and immunoreactive (ir-) inhibin levels in two captive male belugas ("Nack", originally from Canada and "Duke", from the Okhotsk Sea) were measured every month for 9 years between 1995 and 2003. Assuming that clearly increased testosterone levels in the circulation indicates that the belugas had reached sexual maturity, at the ages of 10 ("Nack") and 11 years old ("Duke"). Their testosterone levels before the significant increase (pre-pubertal) were 0.42 ± 0.07 ng/ml (n=18) and 0.35 ± 0.10 ng/ml (n=18) and, those of after the increase (maturity) were 1.65 ± 0.14 ng/m l (n=74) and 2.06 ± 0.14 ng/ml (n=74). Circulating ir-inhibin levels before sexual maturity were 0.78 ± 0.04 ng/ml (n=18) and 0.64 ± 0.04 ng/ml (n=15) and, after sexual maturity were 0.52 ± 0.02 ng/ml (n=56) and 0.43 ± 0.02 ng/ml (n=67). Seasonal changes were observed in the testosterone levels after sexual maturity and the levels increased during March and April in Canadian origin "Nack", and peaked in February in Okhotsk origin "Duke". Circulating ir-inhibin level gradually decreased as they aged. A negative correlation between the circulating testosterone and ir-inhibin was observed. No seasonal changes were observed in the ir-inhibin levels after sexual maturity. These data will surely correspond to clarification of endocrinology and the successful reproduction of the beluga.     

The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.     

Seasonal changes in immunoreactivity of activin signaling component proteins in wild ground squirrel testes

The seasonal spermatogenesis and localization of inhibin/activin subunits (alpha, betaA, betaB) in the testes of wild ground squirrel has been previously described; however, the expression pattern of activin receptors and cytoplasmic signaling SMADs has not been detected in any seasonal breeders. The objective of this study was to investigate the abundance and cellular localization of activin signaling components in testes of the wild ground squirrel during the breeding and nonbreeding seasons. The immunolocalizations of ActRIIB (activin type II receptor B) and activin-related SMADs (phospho-SMAD2/3, SMAD4 and SMAD7) were observed by immunohistochemistry. Total proteins were extracted from testicular tissues in the breeding and nonbreeding seasons and were used for Western blotting analysis for ActRIIB and SMADs. Immunoreactivities of activin signaling components were greater in the testes of the breeding season, and then decreased to a relatively low level in the nonbreeding season. ActRIIB and related SMADs were widely spread in the active testes, while spermatogonia were the predominant cellular sites of activin signal transduction during arrested spermatogenesis. The dynamic regulation of activin type II receptor and SMADs indicated that the activin signal pathway played an important paracrine role in seasonal spermatogenesis of the wild ground squirrel. Furthermore, the distinct localizations and immunoreactivity of ActRIIB and SMADs might suggest different functions of activin in seasonal spermatogenesis.     

Pathological Findings in a Case of Equine T-Cell Lymphoma Associated with Ataxia

A 15-year-old Dutch Warmblood gelding suddenly developed incoordination and hindlimb stumbling. The horse had a history of eyelid lymphoma. Necropsy revealed yellow-white or dark reddish-brown masses adhering to the outer surface of the spinal dura mater from the first cervical vertebra to the seventh thoracic vertebra. The spinal cord close to the first cervical vertebra and the seventh thoracic vertebra was markedly compressed by the masses filling the epidural space. The masses were also observed in the larynx, eyelids, and adipose-rich tissues, including the joints and orbits. They appeared similar in shape. The mandibular, retropharyngeal, axillary, superficial inguinal, deep inguinal, and lateral iliac lymph nodes were solid and enlarged. Histologically, the masses were composed of small or medium-sized lymphocyte-like tumor cells, but atypical cells and mitotic figures were rare. There were moderate infiltrations of macrophages and multinucleated giant cells, which were occasionally ingesting the surrounding tumor cells. Immunohistochemically, the tumor cells were classified as T-cell-derived cells. Throughout the spinal cord, enlargement or loss of nerve axons, dilation of periaxonal spaces, and macrophage infiltration into periaxonal spaces were observed, mainly in the ventral funiculus. Spinal cord compression by the tumor mass was suggested as a cause of the locomotive dysfunction. This is the first report of equine lymphoma with ataxia located from the proximal cervical to middle thoracic dura mater and in joint cavities.     

Active immunization against inhibin improves superovulatory response to exogenous FSH in cattle

The effect of active immunization against inhibin on the response to superovulatory treatment by porcine FSH (pFSH) was investigated in cattle. Japanese black cows were sc injected with 1 mg of porcine inhibin alpha-subunit fragment (1-26) conjugated with rabbit serum albumin (inhibin-immunized group; n=14) or rabbit serum albumin alone (control group; n=12) in Freund's complete adjuvant. Booster injections (half the amount of the primary injection) were given 35 and 70 days after the primary injection. All cows were superovulated three times with pFSH. Three days after each injection of the antigen, a progesterone-releasing intravaginal device (CIDR-B) was inserted vaginally into all animals and left in place for 10 days. Forty-eight hours before CIDR-B removal, all animals were sc injected with 30 mg pFSH dissolved in 40% polyvinylpyrrolidone, and im injected with 750 microg of PGF2alpha at CIDR-B removal. Cows were artificially inseminated twice during estrus, and ova or embryos were collected 7 or 8 days after estrus. The number of corpora lutea, the number of ova or embryos and the number of transferable embryos in inhibin-immunized cows (12.1+/-1.2, 11.1+/-1.3 and 6.2+/-1.0, respectively) were significantly greater than those in the controls (8.2+/-1.0, 5.7+/-1.1 and 3.1+/-0.7, respectively). These results indicate that active immunization against inhibin enhanced ovarian response to the usual superovulatory treatment in cattle. Therefore, immunization against inhibin may be a useful approach for improving the response to superovulation in cattle.     

Reduction of primordial follicles caused by maternal treatment with busulfan promotes endometrial adenocarcinoma development in donryu rats

Ovarian dysfunction leading to hormonal imbalance plays a crucial role in uterine carcinogenesis in rats as well as women. However, the effects of a reduction in primordial follicles at birth on uterine adenocarcinoma development have hitherto not been determined. The present study was therefore conducted using female Donryu rats, a high incidence rat strain of uterine adenocarcinoma. The animals were maternally exposed to 2.5 or 5.0 mg/kg of busulfan on gestation day 14 to reduce primordial follicles, and were then initiated by intrauterine treatment with N-ethyl-N'-nitro-N-nitrosoguanidine at 11 weeks of age. Both busulfan treatment doses caused earlier occurrence of persistent estrus, with dose-dependence as compared to controls. At 15 months of age, the rats were euthanized. The incidence of uterine adenocarcinomas and multiplicity of uterine neoplastic lesions were significantly increased by the 5.0 mg/kg, but not the 2.5 mg/kg busulfan treatment. Morphologically, the ovaries exposed to busulfan treatment exhibited severe atrophy, with few or no follicles and corpus lutea. Serum 17beta-estradiol (E2), progesterone, and inhibin levels were significantly decreased in the busulfan treatment groups, with a clear dose-relation. Interestingly, only the 5.0 mg/kg busulfan treatment elevated the E2/progesterone ratio. These results provide evidence that the reduction of primordial follicles promotes uterine adenocarcinoma development in rats in association with an earlier occurrence of the persistent estrus status.     

Distribution and persistence of emamectin benzoate at efficacious concentrations in pine tissues after injection of a liquid formulation

In an earlier paper the authors reported the creation of a novel emamectin benzoate 40 g litre(-1) liquid formulation (Shot Wan Liquid Formulation). The injection of this formulation exerted a preventative effect against the pine wilt disease caused by the pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, and this effect lasted for at least 3 years. The present study was carried out to show experimentally that the marked effect of this formulation was due to the presence and persistence in pine tissues of sufficient amounts of emamectin benzoate to inhibit nematode propagation. A cleanup procedure prior to quantitative analysis of emamectin benzoate by fluorescence HPLC was devised. The presence of the compound in concentrations sufficient to inhibit nematode propagation in the shoots of current growth and its persistence for 3 years explained the marked preventative effect. Non-distribution of emamectin benzoate in some parts of the lower trunk suggested that the formulation should be injected at several points for large trees in order to distribute the compound uniformly to lower branches.     

A gross and histopathological study of an ectopic white line development in equine laminitis

In horses with chronic laminitis, an abnormal horny structure called lamellar wedge, is generated between the hoof wall and the laminar epidermis. To be able to manage horses with chronic laminitis correctly, more information about the pathological state of this abnormal horn is required. The aim of this study was to collect and analyze objective morphological data about the abnormal horn in order to understand its morphology and development. In the study, the abnormal horn was grossly visible on the sagittal hoof section from approximately 20 days after the onset of disease. In the histological observations, the structural characteristics of this abnormal horn were similar to the white line tissue, suggesting it is an ectopic white line. Mean value of the cross-sectional area of the abnormal horn against the distal phalanx section area (A/D) was 0.29 cm(2) SD +/- 0.14 and it finally showed an eight-fold increase over the mean value of normal white line section area against the distal phalanx section area. In conclusion, a large amount of the ectopic white line is thought to be finally able to inhibit normal hoof wall growth, so that it should be resected at the optimum time when would be after one month from the onset of the disease.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.     

The influence of Pueraria mirifica herb containing phytoestrogens on the urinary gonadotropin and estradiol levels in aged menopausal monkeys

We investigated a non‐invasive method of specimen collection for determining the changes of reproductive hormones in aged menopausal monkeys after a long‐term feeding of the Thai herb Pueraria mirifica (PM) containing phytoestrogens. Three groups of aged menopausal monkeys (n = three in each group) were fed daily with 10, 100, or 1000 mg of PM for a 90 day treatment period, and fed with distilled water for 30 and 60 days of the pre‐ and post‐treatment periods, respectively. Urine samples were collected for 14 h daily every 5 days and assayed for follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol levels. The result showed that monkeys fed with PM 10, PM 100, and PM 1000 had a decrease in urinary FSH levels during the treatment period, followed by a rebound increase during the post‐treatment period. Urinary estradiol levels tended to decrease and fluctuated between 4.28 and 266.71, 2.85–42.27, and 6.24–203.50% of the pre‐treatment levels in those three groups, respectively. Decreases in urinary LH levels could not be observed in all the three groups. These results suggest that FSH could be a candidate marker to detect the estrogenic effects of phytoestrogens in aged menopausal monkeys when changes of urinary hormones need to be used as an indicator.