Ethanol exposure is known to suppress male reproductive activity in laboratory animals and humans. The present study was designed to evaluate whether chronic ethanol exposure decreases proliferative activity or increases apoptosis in the testes. Ethanol (1.5 g/kg or 3 g/kg i.p., 15% v/v in saline) was administrated to adult male rats for 10 days. Proliferating cell nuclear antigen (PCNA) was used as a proliferative marker. Western blot analysis showed that ethanol administration significantly reduced the level of PCNA. Also, immunoreactivity of PCNA-positive cells in the spermatogonia and primary spermatocytes were decreased by ethanol exposure. However, the number of TUNEL-positive cells was significantly increased in the testicular germ cells of ethanol-treated rats. Moreover, ethanol administration significantly increased the level of activated caspase-3 in testes. In conclusion, our findings suggest that ethanol may partly contribute to the suppression of male reproductive activity through a reduction of cell proliferation and an enhancement of cell death in rat testes.

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Estradiol acts as a neuroprotective factor against ischemic brain injury. This study investigated whether estradiol modulates neuroprotective mechanism through the activation of Akt and its downstream target such as Bad in global ischemic injury. Adult female gerbils were ovariectomized and treated with estradiol prior to ischemic injury. Transient cerebral ischemia was accomplished by bilateral clipping of the common carotid artery for 5 min. Brains were collected on 1, 3, 5 day after injury. In hippocampal CA1 region of non-treated gerbils, most of neuronal cells exhibited pyknotic nuclei and showed the positive reaction of TUNEL staining on 5 day after injury. However, estradiol significantly reduced the neuronal cell death. Potential activation was measured by phosphorylation of Akt at Ser⁴⁷³ and Bad at Ser¹³⁶ using western blot analysis. The levels of pAkt and pBad were significantly decreased in non-treated gerbils on 1-5 day after injury. However, estradiol prevents the global ischemic injury-induced decrease of pAkt and pBad. Our findings suggest that estradiol prevents cell death due to global ischemic injury and that Akt activation and Bad phosphorylation by estradiol mediated these protective effects.

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To clarify the relationship between M cells and intestinal microflora, histoplanimetrical investigation into the bacterial colonization and the differentiation to M cells was carried out in rat Peyer's patch under physiological conditions. The follicle-associated epithelium (FAE), except for the narrow area of apical region, was closely covered with both neighboring intestinal villi and a thick mucous layer, the latter of which also filled the intervillous spaces as well as the space between the FAE and the neighboring intestinal villi. Indigenous bacteria adhered almost constantly to the narrow areas of apical regions of both intestinal villi and the FAE. Bacterial colonies were occasionally located on the basal to middle region of FAE, where M cells also appeared, forming large pockets. When bacterial colonies were located on the basal to middle region of FAE, bacteria with the same morphological characteristics also proliferated in the intervillous spaces neighboring the Peyer's patch. In cases with no bacterial colonies on the basal to middle region of FAE, however, M cells were rare in the FAE. Histoplanimetrical analysis showed the similar distribution pattern of bacterial colonies on the FAE and M cells in the FAE. M cells ultrastructurally engulfed indigenous bacteria, which were then transported to the pockets. These results suggest that indigenous bacterial colonization on the FAE stimulates the differentiation of M cells in the FAE under physiological conditions. The uptake of bacteria by M cells might contribute the regulation of the development of indigenous bacterial colonies in the small intestine.

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The glycogen body (GB) is in the dorsal area of the lumbosacral spinal cord in birds and is composed of uniform cells characterized by high glycogen storage. The glycogen of GB cells remains unchanged in vivo by the effects of a variety of hormones such as insulin, glucagon, adrenocorticotropic hormone and by physiological conditions such as starvation. In order to investigate the latent functionability of GB cells, we observed morphological changes of glycogen body cells in a co-culture system with cerebellar neurons by light and transmission electron microscopy. Cultured GB cells were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). The cultured neurons derived from cerebellum were co-cultured with the labeled GB cells. Under the co-culture with neurons, 2 types of GB cells were detected. One was conventional with numerous glycogen deposits in the cytoplasm and tended to make clusters. The other type of GB cells singly extended the processes attaching to the neuronal body and axons. In the axons in contact with GB cell processes, small vesicles appearing as synaptic vesicles were observed. These observations suggested that some GB cells can differentiate to an average astrocyte. The GB cells were assumed to involve the synapse formation or maturing as astrocytes in the CNS.

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Serum obtained from a patient histopathologically diagnosed as intestinal spirochetosis was investigated serodiagnostically by agglutination test. B. aalborgi which is a human intestinal spirochete reacted strongly with the human serum, while B. pilosicoli which has potential pathogenicity to humans reacted with the serum, but as strongly and its titer was different than the other three species. On the other hand, intestinal spirochetes (Matsumoto isolates) were isolated from the biopsy samples of the patient. The morphological, biochemical, and genetic characteristics of the isolates were very similar to those of B. aalborgi. Furthermore, the protein profiles of the Matsumoto isolates were also similar to those of B. aalborgi but were different than those of B. pilosicoli and B. hyodysenteriae. The reaction profiles of the Matsumoto isolates in immunoblotting were relatively similar to those of B. aalborgi except for a 74 kDa band but were different from those of B. pilosicoli and B. hyodysenteriae. Therefore, we identified the Matsumoto isolates as B. aalborgi and diagnosed the patient with a B. aalborgi infection.

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We investigated the susceptibilities against 7 antimicrobial agents in Campylobacter jejuni and C. coli isolates from food-producing animals in 2004. In comparison with the results of past surveillance, no significant difference was observed in resistance rates against all of the antimicrobials tested in Campylobacter isolates. However, slight increase of erythromycin (EM) resistance was found in C. coli isolates from pigs. We examined the mutation of the 23S rRNA gene and their susceptibilities against azithromycin, tylosin, and lincomycin in 44 EM-resistant isolates and 28 susceptible isolates of porcine origin. All the EM-resistant isolates contained A2075G in the 23S rRNA gene and showed cross-resistance to azithromycin, tylosin, and lyncomycin.

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Brucella, a causative agent of brucellosis and facultative intracellular pathogen, has been isolated recently from a variety of wild mammals. In this study, serum samples from 115 Japanese wild boar (Sus Scrofa leucomystax) killed by hunters in the 4 Prefectures of Shikoku, Japan were tested for antibodies to Brucella spp. by means of the tube agglutination test (TAT) and enzyme-linked immunosorbent assay (ELISA) using antigens extracted with n-lauroylsarcosine. In 9 of the 115 samples (7.8%) antibodies to Brucella spp. were detected by TAT and ELISA. These results suggest that wild boar in Shikoku may be exposed to Brucella spp. or other cross-reactive pathogen infection.

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Genetically modified corn Bt11 is insect-resistant and expresses Cry1Ab toxin, an insecticidal protein, in kernels. Although Bt11 corn is considered safe based on animal performance, there are no reports available on the clinico-biochemical effects of feeding it to cattle. In this study, we evaluated the effects of feeding Bt11 to calves, using blood and ruminal clinico-biochemical parameters. Our three-month-long feeding experiment demonstrated that calves (n=6), fed with a ration containing 43.3% of Bt11 corn kernels as dry matter, did not develop any discernible clinical, hematological, biochemical, or ruminal abnormalities as compared with control calves (n=6) fed non-Bt11 corn. The results suggest that the transgenic Bt11 has no negative clinico-biochemical effects on calves.

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To investigate the effects of oral administration of an interferon (IFN)-α drug on the immune reaction of healthy Japanese Black (JB) calves, peripheral leukocyte populations and their ability to produce cytokine mRNA were analyzed after oral administration of IFN-α. Fourteen calves fed in one herd were divided into two groups; seven calves were orally administered 0.1 g/day of IFN-α from the day of birth to day 5 on each day (group 1, N=7), and the other seven calves were used as the control (group 2, N=7). Blood samples were collected from the jugular veins of all calves before administration and in weeks 1, 2, 4, 8, and 16 after birth. The number of MHC class II⁺CD14⁺ monocytes in the leukocytes population of group 1 increased gradually after birth, and significantly higher numbers were detected in week 4 compared with group 2. MHC class II^-CD14⁺ monocytes in group 1 peaked in week 1, and a significant increase was detected compared with group 2. The level of IL-12 in the cytokine mRNA of group 1 increased gradually between weeks 1 and 2, and a significantly higher level of IL-12 was found compared with group 2. These results suggest that oral administration of IFN-α induces activation of the monocyte functions in JB calves.

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A dog histopathologically diagnosed with hepatocellular carcinoma (HCC) showed very high serum alkaline phosphatase (ALP) activity. A supernatant of ascitic fluid and tumor tissue extracted from the dog also showed much higher ALP activity than normal. ALP isoenzyme analysis of samples was perfomed using polyacrylamide gel disk electrophoresis, and a wide, broad abnormal band was observed. By various treatments, the abnormal band showed thermostability, which is a characteristic of tumor-associated ALP that has only been reported in humans. The thermostable ALP isoenzyme was not found in sera from 39 dogs with several types of tumor that originated from the liver, except for HCC, nor was it found in 10 dogs with hepatic diseases that did not include hepatic tumors. The thermostable ALP isoenzyme seemed to be associated with canine HCC.

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Toxoplasma gondii from pigs in Okinawa Prefecture was characterized by nested PCR-restriction fragment length polymorphism (RFLP) and DNA sequence analysis of the dense granule antigen GRA6 gene. By nested PCR, parasite DNA was detected in 33 out of 91 lymph node samples with lesions similar to those found in toxoplasmosis samples that had been collected from pigs at an abattoir. RFLP analysis with MseI was successfully conducted in 29 of 33 PCR-positive samples to group the isolates into one of the three genotypes of T. gondii. Genotyping of the 29 studied samples rendered the following results: 13 of type I (44.8%), 14 of type II (48.3%), and 2 of type III (6.9%). The GRA6 genes of 12 Okinawa isolates were cloned and sequenced. Nine new nucleotide sequences were found, and nucleotide substitutions specific for the Okinawa isolates were found at 13 positions. Phylogenetic analysis indicated that all GRA6 sequences were divided into one of the 3 main groups, and Okinawa isolates of GRA6 genotypes II and III seemed to be closely related to the Beverley strain and the NED strain, respectively. The results from this study may provide basic and useful information for the analysis of the molecular epidemiology of T. gondii infection within Japan.

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A cell line, MCO-Y4, was established from a mammary gland osteosarcoma of a 16-year-old female mongrel dog. Histopathologically the tumor was composed of osteoblastic cells with an osteoid meshwork and chondroid matrix. The mean doubling time of the cells at the 93rd passage was 32.39 ± 4.66 hr. Immunohistochemically, the osteoblastic and chondroblastic cells were positive for bone morphogenetic protein (BMP)-2/4 and BMP receptor (BMPR) II. The cultured cells were spindle in shape during the growth and the confluent phases. No tumor matrix was detected in the culture dish by alcian blue staining or von-Kossa silver impregnation. MCO-Y4 cells on the chamber slides showed intense immunoreactivity for BMP-2/4 and BMPR II. Noggin, an antagonist for BMP-2/4, showed the growth inhibition on MCO-Y4 cells. In addition, fibronectin might be potential for stimulating growth of MCO-Y4 cells. When transplanted into severe combined immunodeficiency mice, the cells formed tumors consisting of solid proliferation of osteoblastic and fibroblastic cells with woven-bone trabeculae. These tumor cells were intensely positive for BMP-2/4 and BMPR II. Our results suggested that the cell line might be useful for studying the role of BMPs in canine osteosarcoma and the mechanism of ossification.

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A 4-year-old male Shiba dog initially presented with pain of an undetermined origin and hypersensitivity to touch. Seven days later, the dog developed ataxia, hind-leg weakness and knuckling. The dog died on 20 days after presentation. Postmortem examination revealed a mass in the body of thoracic vertebra. Histopathologically, the mass consisted of granulomatous inflammation, including fungal organisms that were immunohistochemically positive for Candida albicans. Similar granulomatous lesions were observed in the systemic lymph nodes, kidneys, pancreas, spleen, prostate gland, thyroid glands and heart. This case was diagnosed as systemic candidiasis with spondylitis.

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A high-K⁺, Na⁺-deficient (I-154 K⁺) solution induced contraction followed by gradual relaxation of the smooth muscles of the bovine trachea, while hyperosmotic addition of 65 mM KCl induced a large sustained contraction. Exposure of the muscle to the I-154 K⁺ solution induced an increase in the ratio of cellular water content and a sustained increase in oxidized flavoprotein fluorescence or reduced pyridine nucleotide fluorescence. The I-154 K⁺ solution also induced a sustained increase in [Ca²⁺]i level. Decreases in developed tension and increases in cellular water content were both prevented by the addition of sucrose or NaCl but not pyruvate. Substitution of KI for KCl in the I-154 K⁺ solution produced a greater inhibition of contraction, while substitution with K-propionate produced no inhibition of contraction. Moreover, decreases in developed tension and increases in cellular water content were both prevented by addition of 100 μM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), but not 10 μM bumetanide or 1 mM acetazolamide. In conclusion, I-154 K⁺ solution induced-relaxation in the bovine trachea may be due to swelling of smooth muscle cells and the mechanism of swelling is probably involved in DIDS-sensitive anion movement.

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The effects of melatonin (MEL) injection into the third ventricle (3V) on growth hormone (GH) secretion were investigated in conscious Holstein steers. A stainless steel cannula was stereotaxically implanted in the 3V based on the ventriculogram. In Exp. 1, three doses of MEL (100, 300 or 600 μg) were injected into the 3V through the cannula and the GH concentration after the injection was determined. In Exp. 2, intracerebroventricular (icv) and intravenous (iv) injections of MEL (100 μg) and GH-releasing hormone (GHRH; 0.25 μg/kg body weight), respectively, were performed simultaneously to examine the effect of MEL on GHRH-induced GH release. The icv injection of MEL significantly stimulated GH release at 100 μg. The increase in GH concentrations by 100 μg of MEL was persistent. Intravenous injection of GHRH dramatically increased GH release. The injection of MEL did not alter GHRH-induced GH release. These results suggest that MEL stimulates GH secretion possibly through the hypothalamus in cattle.

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A brachypodism (brp) mutation arose spontaneously in the inbred NC mouse strain, producing a phenotype similar to that caused by bp mutation; therefore, it is strongly suggested that brp and bp are allelic. A series of bp mutations are due to defects in the growth differentiation factor 5 (Gdf5) gene. Nucleotide sequence analysis on the Gdf5 gene in NC-brp/brp mice revealed that an irregular insertion of a unit `GGCAGCC' in exon 2 caused a frame shift leading to a premature stop codon. In addition to the known physiologic roles of brp, I found that brp significantly reduced the litter size. The brp is a novel mutant allele at the Gdf5 gene locus; I would like to name this allele Gdf5^brp.

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The freezing conditions for preparation of frozen canine semen by the plunging method were investigated with regard to the period of sensitization in liquid nitrogen (LN₂) vapor and the height from LN₂, and the semen qualities after thawing were compared with those of canine semen prepared by the simple freezer method previously reported by us. In the plunging method, 9 semen straws were prepared under the same conditions, horizontally kept at 5, 7, and 10 cm above the LN₂ surface in a styrene foam box for 5, 10, and 15 min, and then plunged into LN₂. The semen qualities immediately after thawing were high in the 7 cm/10 min (cooling rate: -4 to -22°C/min) and 10 cm/15 min groups (cooling rate: -6 to -10°C/min). On comparison of frozen semen prepared by the plunging method (7 cm/10 min) with frozen semen prepared by the simple freezer method, sperm motility and viability were significantly higher for the frozen semen prepared by the plunging method. The cooling rate in freezing was higher for the simple freezer method (cooling rate: -6 to -50.9°C/min) than the plunging method. Based on these findings, horizontal placement of canine semen straws above LN₂ to reduce the temperature at a slow cooling rate of about -10°C/min, followed by plunging into LN₂ after sensitization for 10-15 min, provides good semen qualities after thawing.

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We fabricated a prototype 3.25-MHz split-focus therapeutic transducer combined with a small 6.5-MHz imaging ultrasonic probe for transrectal treatment of prostate cancer and evaluated the feasibility of using split-focus high-intensity focused ultrasound (HIFU) to ablate localized tumor tissue without injuring the surrounding organs. We therefore established a localized tumor model by inoculating VX2 tumor into rabbit livers. The localized VX2 tumors of nine rabbits were transdermally treated with split-focus ablation at a peak intensity in water of 6 kW/cm² for 4 s (6 shots) under the guidance of ultrasonic B-mode imaging. Necropsy a day after treatment found the surface of the livers and gastrointestinal tracts to be grossly normal. The VX2 tumors were completely coagulated and were surrounded by ablated liver tissue. The six shots of split-focus HIFU destroyed the VX2 tumors without injuring the liver surfaces or the surrounding organs. These results suggest that split-focus HIFU ablation could be an effective treatment of localized tumors.

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The serum and fecal testosterone (T) concentrations and testicular sizes of two male bharals (Pseudois nayaur) were determined for approximately one year. The profiles of the fecal T concentrations showed a similar tendency as the profiles of serum T concentrations, and there was a significant correlation between serum and fecal T concentrations (r=0.72). T concentrations rose drastically in October and decreased gradually until January. The maximum testicular size was observed between November and January. Semen collected between December and January was excellent in quality and comparable to domestic sheep and goats. The active periods of the testes were synchronized with the early breeding season of females.

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Seven mature Japanese black bears were used as semen donors, and a total of 7 semen samples collected from the animals by the electroejaculation method were cryopreserved in liquid nitrogen. Egg yolk-TRIS-citrate-glucose extender was used, and the effects of different final concentrations of glycerol, at 4-12% (v/v), on frozen-thawed spermatozoa were examined. No significant difference was observed in percent motility or percent abnormal morphology of frozen-thawed spermatozoa among the different glycerol concentrations. Percent viability and percent intact acrosomes of spermatozoa cryopreserved with 4 and 6% glycerol were significantly higher than those with 10 and 12% glycerol. These results suggest that a suitable glycerol concentration for freezing Japanese black bear semen within the range tested would be 4-6%.

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The addition of Orvus ES paste (OEP) to extender may be essential for preparing frozen dog semen. The major ingredient of OEP is sodium lauryl sulfate (SLS). In this study, we compared the effect of SLS on frozen dog semen with that of OEP. There were no significant differences between the 2-mg/ml SLS group and OEP group concerning sperm motility, viability and the percentage of viable sperm with intact acrosomes after freeze-thawing. These results suggest that the effectiveness of frozen dog semen extender containing 2 mg/ml of SLS is similar effective to that demonstrated for OEP.

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The purpose of this study was to ascertain whether or not Japanese black bears (Ursus thibetanus japonicus) are induced ovulators. The progesterone levels of female bears kept with a male and allowed to mate (n=2) and female bears allowed contact with a male through bars but not allowed to mate (n=6) during the mating season increased significantly in late October. Based on this result, the female bears were considered to have ovulated. Females isolated from males (n=3) were ovarioectomized after the mating season, and no corpora lutea were observed, indicating they had not ovulated. These findings suggest that Japanese black bears may be induced ovulators that ovulate with stimuli from males and without coitus at a high rate.

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To develop a live vaccine for equine herpesvirus type 1 (EHV-1), two EHV-1 mutants containing no heterogeneous DNA, ΔgI and ΔgE, were constructed with deletions in the open reading frame of either glycoprotein I (gI) or E (gE), respectively. In equine cell culture, deletion mutants formed smaller plaques than the parental and revertant viruses, but the one-step growth patterns of the deletion mutants and the parental strain were approximately the same. These results suggest that both gI and gE contribute to the ability of EHV-1 to spread directly from cell-to-cell, but that these glycoproteins are not required for viral growth in vitro. Mice and hamsters inoculated intranasally with these mutants showed no clinical signs, and continued to gain weight, whereas those inoculated with the parental virus exhibited a reduction in mean body weight. Furthermore, nervous manifestations were observed in hamsters inoculated with the parental virus. These results suggest that gI and gE have an important role in EHV-1 virulence including neurovirulence in experimental animal models. On the other hand, serum neutralizing antibodies were detected in mice immunized with ΔgI or ΔgE at two weeks after inoculation. Following challenge with the parental virus, ΔgI- or ΔgE-immunized mice were able to clear parental virus from their lungs faster than mock-immunized mice. These results suggest that the EHV-1 mutants defective in gI and in gE are attenuated but have ability to elicit immune responses in inoculated mice that contribute to virus clearance.

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Seventy-seven rabies virus (RV) isolates originating from Brazilian cattle were genetically characterized. Partial nucleoprotein gene sequences of these isolates were phylogenetically and geographically analyzed. Cattle isolates, which clustered with the vampire bat-related RV group, were further subdivided into nine genetic subgroups. These subgroups were distributed widely in lowland regions, with some subgroups separated from each other by mountain ranges. In addition, separation of the groups in mountainous regions was correlated with altitude. These results indicate that cattle rabies is derived from several regionally-defined variants, which suggests that its geographical distribution is related to that of the vampire bat population.

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