Immunolocalization of steroidogenic enzymes P450scc, 3betaHSD, P450c17, and P450arom in Göttingen miniature pig testes

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in G?ttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.     

Formulation of low phosphorus loading diets for carp (Cyprinus carpio L.)

This study focuses on reducing total phosphorus loading (T-P) from carp culture through improved feed formulation. Since phosphorus (P) contained in fish meal (FM) mainly in the form of tricalcium phosphate is not available to carp, which lack a stomach, the reduction of FM from their diets is effective for lowering T-P. Thus in this experiment, six diets (crude protein < 35%, digestible energy > 3.5 kcal g⁻¹) were designed by substituting FM (10%−25%) with alternate protein ingredients such as poultry feather meal (PFM; 5%−10%), blood meal (BM; 5%−7%) and defatted soybean meal (dSBM; 4%−9%). All diets followed the Kasumigaura ‘Feed Standard’. The total dietary P was 1.0%−1.4% and water extractable P available to carp was 0.66%−0.71%, the levels meeting the dietary requirement of carp. A feeding trial was conducted with juvenile carp (4.6 ± 0.7 g) for 12 weeks at a mean water temperature of 23.7°C. The T-P loading from fish fed the different diets was estimated based on absorption and retention of dietary P. Growth performance corresponded to increasing levels of FM inclusion, being highest in the fish fed 25% FM diet; however, the decrease in T-P was achieved at the lower FM levels. The T-P (based on P retention) ranged from 8.9 to 11.7 kg t⁻¹ production, much lower than that from the commercial diets (9.1–26.4 kg t⁻¹ production). These results indicated that the reduction of FM levels in carp diets to 15%−20% through the combined use of PFM, BM and dSBM effectively lowered T-P. Moreover, the formulated diets were also found to be better than commercial diets in lowering the N loading from carp culture.     

Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus

Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed.     

High prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 from cattle in selected regions of Japan

The prevalence of enterohemorrhagic Escherichia coli (EHEC) O157 was examined in bovine faeces. EHEC O157 was isolated from the faeces of 42 (13.0%) of 324 cattle. Of the 4 farms and the facilities tested, the 3 farms and the facilities were found positive for EHEC O157. The highest isolation rate among the farms was 33.7%. The prevalence of EHEC O157 in heifers was higher than that in calves and other cattle. No cattle positive for EHEC O157 showed any clinical signs except 2 calves with diarrhea in a veterinary hospital. Almost all isolates possessed the stx gene, and Stx-positive strains carrying both stx(1) and stx(2) genes were predominant. These results indicate that EHEC O157 are distributed in bovine faeces, and that dairy and beef farms in selected regions of Japan are heavily contaminated with the organisms.     

Passive immunoneutralization of endogenous inhibin increases ovulation rate in miniature Shiba goats

We reported previously that passive immunization against inhibin enhances follicular growth and increases the ovulation rate. However, the ovulation rate was not comparable to the number of follicles. Therefore, the aim of this study was to attempt to increase the ovulation rate by increasing the interval between inhibin immunization and PGF2alpha injection. Five miniature Shiba goats were treated with 10 ml inhibin antiserum (inhibin-AS) developed against [Tyro30]-inhibin alpha (1-30). A control group (n=5) was treated with normal goat serum. All animals were injected intramuscularly with 125 microg PGF2alpha 72 h after treatment to induce estrus and ovulation. Blood samples were collected for hormonal assay and the ovulation rate was determined by laparotomy. In contrast to the control group, there was a significant increase in plasma concentrations of FSH in the immunized group. After luteolysis, plasma concentrations of estradiol-17beta increased markedly to a preovulatory peak about 2 folds higher (P<0.01) than that of controls. In addition, the ovulation rate was greater in the immunized group (14.4 +/- 2.2) than in the control group (2.2 +/- 0.6), and the mean number of follicles > or = 4 mm in diameter was 10.0 +/- 0.8 in the inhibin-AS group compared with 2.4 +/- 0.3 in control group. The present results demonstrate that immunoneutralization of endogenous inhibin increased FSH secretions in miniature shiba goats. The increased FSH secretion enhanced follicular growth and increased the ovulation rate. Additionally, increasing the interval between inhibin-AS and PGF2alpha injections (to 72 h) resulted in a greater ovulation rate compared with the previous protocol (48 h). Therefore, inhibin-AS treatment proved to be an effective alternative to exogenous gonadotropin methods for induction of superovulation in goats.     

Expression of nerve growth factor (NGF), and its receptors trkA and p75 in ovaries of the cyclic golden hamster (Mesocricetus auratus) and the regulation of their production by luteinizing hormone

In the present study, changes in localization of nerve growth factor (NGF) and its receptors, trkA and p75 in the ovary were investigated during the estrous cycle of the golden hamster. The effect of LH surge on changes in localization of NGF, trkA and p75 in the ovary was also investigated. NGF and its receptors trkA and p75 were localized in oocytes, granulosa cells and theca cells of various stages of follicles throughout the estrous cycle. NGF and its two receptors were also present in numerous interstitial cells and luteal cells. The number of interstitial cells staining positively for NGF and its two receptors was greater in ovaries of day 1 (day 1=day of ovulation) than the other days during the estrous cycle. Treatment with the antiserum against luteinizing hormone releasing hormone (LHRH-AS) at 1100 h on day 4 completely blocked ovulation. There were few positive reactions for NGF and its two receptors in interstitial cells 24 hr after LHRH-AS injection. The effect of LHRH-AS treatment was blocked by a single injection of 10 IU human chorionic gonadotropin. The distinct widespread distribution of NGF and its two receptors in the ovary of golden hamsters suggest that NGF may be an important growth factor for regulation of ovarian function. Furthermore, the LH surge may be an important factor for inducing production of NGF and its two receptors in interstitial cells of the cyclic golden hamster.     

Characterization of the chicken PKR: polymorphism of the gene and antiviral activity against vesicular stomatitis virus

The double-stranded RNA-dependent protein kinase (PKR) is induced in mouse and human cells on treatment with interferon. In this study, we have cloned and determined the nucleotide sequences of chicken PKR cDNA in various chicken breeds. Chicken PKR was a 550-amino-acid protein as deduced from the cDNA open reading frame (ORF), and there were specific domains (two double-stranded RNA binding domains (DRBDs) and numerous kinase subdomains) characterized in RNA binding proteins and kinase families. Furthermore, it was suggested that chicken PKR was polymorphic. Transfected cell clones expressing chicken PKR mRNA were demonstrated to confer antiviral responses to vesicular stomatitis virus, except for Koshamo type-3 (KS-3). KS-3 PKR, which has an amino acid substitution at position 507 (Arg to Gln), showed amphibious antiviral responses. This specific amino acid substitution was considered to determine the antiviral function of chicken PKR in addition to essential domains as DRBDs and kinase subdomains.     

Early pregnancy diagnosis by means of ultrasonography as a method of improving reproductive efficiency in goats

Eighteen cyclic Shiba goats were used in this study. Estrus was synchronized with a single injection of 125 microg of a synthetic analogue of prostaglandin F(2)alpha (PGF(2)alpha) after detection of at least one corpus luteum by B-mode ultrasonography. Blood samples were collected from each animal on days 0, 7 and 21 post-mating for progesterone assay. Animals in estrus were either allowed to be mated by fertile bucks twice during estrus (group I; n=12) or not at all (group II; n=6). Ultrasonographic examinations were performed transrectally or transabdominally using a real-time B-mode scanner equipped with a 7.5 or 5 MHz transducer. All animals exhibited estrus 56.0 +/- 2.7 h after injection of PGF2alpha. The results show that the accuracy of the progesterone assay in diagnosing pregnancy on day 21 after mating was 80% for pregnancy and 100% for non-pregnancy, retrospectively. Ultrasonographic examinations showed that gestational sac and embryos heartbeats were detected on days 20.2 +/- 0.6 and 24.3 +/- 0.7 of gestation, respectively. Placentomes were detected on day 35.4 +/- 1.0 of gestation as small nodules (0.7 +/- 0.2 cm in size). At two months pregnancy, skeletal structures like skull, thorax and long bones were clear. Biparietal diameter of the skull and length of long bones could be used as an estimate of gestational age. The accuracy of detection of fetal number using real-time B-mode ultrasonography was 91.7% on day 60 of gestation. In conclusion, progesterone assay at day 21 post-mating (cut-off value, 1 ng/ml) can be used for pregnancy diagnosis in goats. However, B-mode transrectal ultrasonography was more efficient due to detection of embryo and confirmation of its viability by heartbeats. In addition, fetal number and gestational age could be determined only by ultrasonography.     

Possible vector insect of mulberry dwarf phytoplasma, Tautoneura mori Matsumura

Mulberry dwarf (MD) phytoplasma was surveyed for its presence in presumably nonvector insects (two species of leafhoppers, thrips, and spider mites) collected from MD-diseased trees. MD phytoplasma was found in all species by nested polymerase chain reaction using specific primers for the 16Sr I-group phytoplasma. By electron microscopy, MD phytoplasma was observed in the salivary glands of the leafhopper Tautoneura mori Matsumura. In addition, the vector ability of T. mori was verified through bioassay; 25% of seedlings inoculated with infective leafhoppers had MD symptoms, suggesting that T. mori may act as a vector of MD phytoplasma in fields in some cases.