Gastrin-releasing peptide (GRP), a mammalian homologue of amphibian bombesin, has been suggested to be a novel regulatory peptide in the reproductive tract during pregnancy. In this study, the localization of GRP in the bovine uterus and placenta was demonstrated by immunohistochemistry. Uterine and placental samples were collected from nonpregnant and pregnant specimens, respectively. Tissue sampling was done from the caruncle and intercaruncle of the uterus, and from the placentome (caruncle and cotyledon) and intercotyledon of the placenta. In all the tissues examined, GRP was detected although its immunoreactivity was observed at various degrees. In the uterus, moderate immunoreactivity for GRP was observed in the uterine gland epithelial cells. In the placenta, strong immunoreactivity for GRP was demonstrated in the uterine gland epithelial cells; moderate in superficial epithelial cells; and weak in the trophoblasts, trophoblastic giant cells and cryptal epithelial hybrid cells. In both nonpregnant and pregnant animals, GRP was immunolocalized in the uterine gland secretions and was found predominantly in the supranuclear region of the uterine gland epithelial cells. These findings may suggest that GRP is secreted into the uterine lumen and regulates the intrauterine environment of both the nonpregnant and pregnant bovine by exocrine, autocrine and/or paracrine manner.

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Spermatogenesis and acrosomal formation in the greater Japanese shrew mole, Urotrichus talpoides, were studied by light microscopy. On the basis of acrosomal changes, morphology of spermatid head, nuclear shape, appearance of meiotic figures, location of spermatid and period of spermiation, the cycle of the seminiferous epithelium was classified into 12 stages, and developing spermatids could be divided into 15 steps. The mean relative frequencies of stages from I to XII were 10.9, 8.7, 9.8, 7.3, 8.5, 10.3, 12.5, 8.7, 5.8, 5.4, 5.1 and 7.1%, respectively. Similar to the case in the musk shrew, the spermatid nucleus of the greater Japanese shrew mole remained in the middle region of the seminiferous epithelium and only the acrosome extended towards the basement membrane. The elongation of the acrosome, however, was not prominent. The proacrosomal vesicle first appeared in stage II and then one large and round granule was seen in stage III. The acrosomal vesicle became flattened on the surface of the nucleus in stage IV. Spreading of the acrosomic system has been recognized from stage VII. In stage VII, spermiation occurred. In stage IX, the spermatid nucleus began to elongate. Elongation and condensation of the nucleus were clearly observed in stage X. In stage XII, pachytene spermatocytes divided into diplotene spermatocytes. In stage XII, meiotic figures and secondary spermatocytes were observed.

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The distribution of reticulocerebellar (RC) neurons was examined by the retrograde transport of horseradish peroxidase (HRP) or wheat germ agglutinin bound to HRP (WGA-HRP) in 7 White Leghorn chickens. A large number of labeled cells were found in the nucleus reticularis pontis caudalis (RP) of the pontomedullary junction and in the nucleus reticularis gigantocellularis (Rgc), parvocellularis (Rpc), subtrigiminalis (Rst), paragigantocellularis (Rpg) and paramedianus (RpaM) in the medulla. Slightly ventral to the vestibulocochlear nerve were many large RC neurons arranged in a longitudinal manner along the lateral edge of the brainstem reticular formation (the Dorsolateral edge cells, DLe cells). RC neurons were most numerous in the Rgc and accounted for 31.9% of the total number of labeled cells, followed by RP (24.2%), Rpc (12.7%), Rpg (10.8%), RpaM (6.7%), DLe cells (6.3%) and Rst (4.9%). The great number of RC neurons was found around the levels of the vestibulocochlear nerve.

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Relationships between female reproductive performance and uterine natural killer (uNK) cells were investigated in pregnant IL-2 receptor β-chain overexpressed transgenic (Tg2R β) mice. At 8 days of pregnancy, all fetuses were alive, suggesting that implantation normally occurred in these mice. However, 47% of fetuses were dead at 10 days of pregnancy and at 12 days all fetuses were resorbing, indicating that fetal loss progressed with the advance of pregnancy. The placenta of Tg2Rβ mice gradually decreased in weight with the advance of pregnancy. At 10 days the placental labyrinth, decidua basalis, and metrial gland in Tg2Rβ mice were poorly developed, and more uNK cells were found in Tg2Rβ mice than in the control mice. We propose that Tg2Rβ mice are the first and interesting model that uNK cells can cause abortion, to clarify the involvement of uNK cell function in female reproductive performance.

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A field trial was conducted to evaluate effect of enrofloxacin-Na against pathogens related to the respiratory and alimentary diseases in eighty suckling piglets (6-7 days old) and eighty weanling piglets (5-6 weeks old). Respective twenty of the suckling and weanling piglets were assigned to each of 4 experimental groups; control (non-treated), clinical injection dose (CID), 2x clinical injection dose (2CID), and premix. A 0.05 ml (2.5 mg) of enrofloxacin-Na injection (5% solution, 1 ml) per kg body weight of piglets as CID was injected intramuscularly for 3 days and the clinical signs were observed for 9 days. The premix (150 ppm) of enrofloxacin-Na was administered with feed for 7 days ad libitum and the clinical signs were observed for 13 days. The enrofloxacin-Na-treated piglets showed a higher increase in body weight and a lower feed per gain than the control piglets. In addition, the treatment of enrofloxacin-Na, regardless of the route of administration, decreased the incidence rate of diarrhea in suckling piglets and respiratory symptoms in weanling piglets. The isolation index of E. coli and Cl. perfringens during the treatment periods was also lowered by the enrofloxacin-Na treatment in both suckling and weanling piglets. The antibiotics was also evaluated as safe locally and whole bodily as treated by injection or feeding. These results indicate that the newly developed antibiotics, enrofloxacin-Na, is very useful for the prevention and therapy of swine diseases in the pig industry.

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A culture condition supporting adipocyte differentiation of stromal-vascular (S-V) cells isolated from canine adipose tissues was established. Morphological observation and determination of glycerol-3-phosphate dehydrogenase (GPDH) activity were used as the criteria for adipocyte differentiation. After reaching confluence, the cells were able to undergo terminal adipocyte differentiation by treatment with 100 μM indomethacin, 10 μg/m l insulin and 0.5 mM 1-methyl-3-isobutylxanthine (MIX) in medium supplemented with 5% fetal calf serum (FCS). In the absence of either indomethacin or insulin, the S-V cells did not undergo adipose conversion and GPDH activity was not increased, indicating that both indomethacin and insulin play essential roles in this culture system. The S-V cells from inguinal adipose tissues exhibited the greatest increase in GPDH activity among the four depots (inguinal > abdominal-subcutaneous > perirenal > omental), demonstrating that adipocyte differentiation was also intensely dependent on anatomic sites from which the S-V cells were derived. Interestingly, dimethylsulfoxide (DMSO) was found to accelerate adipocyte differentiation in combination with indomethacin and insulin. Under this condition, up to 90% of the cells displayed adipocyte phenotypes and the GPDH activity reached 1288 ± 441 mU/mg protein. This culture system may be useful for investigating other adipogenic factors as well as anti-adipogenic factors involved in the regulation of canine adipose tissue development.

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Haptoglobin (Hp) is a hemoglobin (Hb)-binding acute-phase protein. Besides its relevance in inflammation, Hp is involved in the regulation of lipid metabolism. In cattle, in addition to the lipoprotein-deficient fraction, Hp is distributed in high-density lipoprotein (HDL) and very high-density lipoprotein (VHDL) fractions. The purpose of this study was to determine Hp concentrations in the lipoprotein fractions using an enzyme-linked immunosorbent assay (ELISA) based on the affinity with Hb, and also to detect structural differences of HDL Hp from that in the lipoprotein-deficient fraction using 2-dimensional electrophoresis. When purified Hp was used as the antigen for the ELISA, the detection limit was 7.4 ng/ml and linearity was obtained from 14.8 to 475 ng/ml. The correlation coefficient between the ELISA and single radial immunodiffusion was 0.884. The ELISA was shown to be applicable to evaluate Hp concentrations in the lipoprotein fractions. Hp concentrations in the lipoprotein fractions were in the range of 0.94 to 8.77 μg of Hp/ml (n=4), and concentration ratios were 0.2 to 0.3% of whole serum Hp. Of the lipoprotein fractions, Hp was most abundant in HDL, moderate in VHDL and faint in chylomicrons, the very low-density lipoprotein fraction and low-density lipoprotein fraction. By 2-dimensional electrophoresis, α- and β-chains of serum Hp were each separated into 5 spots, and their isoelectric point (pI) values were from 5.05 to 6.28 in the α-chain and from 5.92 to 6.95 in the β-chain. The pI values of HDL Hp were indistinguishable from those of serum Hp. These results indicate that the ELISA based on the affinity with Hb is useful for evaluating Hp concentrations in lipoprotein fractions, and also suggest that HDL Hp is structurally similar to that in the lipoprotein-deficient fraction.

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Apolipoprotein (apo) C-III is a low-molecular-mass protein that is involved in the regulation of the triglyceride metabolism. Except for the hyperlipidemic calf, cattle apoC-III is mainly detected in the high-density lipoprotein (HDL) fraction, and the distribution in chylomicrons (CM) and the very low-density lipoprotein (VLDL) fraction has not yet been clarified. The purpose of the present study was to detect apoC-III in concentrated CM and VLDL fractions to examine whether apoC-III is distributed in the two fractions even in normolipidemic cattle. ApoC-III could be detected by immunoblot analysis in both concentrated cow CM and VLDL fractions, but not in the corresponding calf fractions. These results suggest that apoC-III is distributed in the CM and VLDL fractions, at least in cows, although the concentrations in these fractions are considerably lower than in the HDL fraction.

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Polymerase chain reaction (PCR) was first applied to diagnosis of canine babesiosis in Japan. Blood samples from 13 dogs suffering from canine babesiosis were used for examination of specificity and sensitivity of the PCR diagnosis. Of the 13 dogs, three were experimentally infected, and ten were naturally infected with Babesia species in west part of Japan. We designed a nested PCR to amplify the babesial small subunit ribosomal RNA gene and found that only the nested PCR produced a visual band, which were not apparent by the first-round PCR to the positive samples. Specificity of the nested PCR was confirmed by amplification after the second-round PCR. Sensitivity of the nested PCR was examined by diluting the blood samples from infected and uninfected dogs. The nested PCR was found to show positive results on the most diluted blood at 0.0001% parasitemia. These results indicate that the nested PCR is highly sensitive and useful for diagnosis of canine babesiosis.

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The expression of cytokeratins and involucrin was analyzed to identify the skin cells which compose the epidermis of dogs. The distribution of cytokeratins and involucrin in normal dog skin was immunohistochemically examined with 27 commercial monoclonal antibodies for human use. Antibodies, No.4, OV-TL12/13, 35βH11, 4.1.18, CAM5.2, NCL5D3, Ks.13.1, Ks.18.04, Ks.19.1, 170.2.14 and Ks.20.8 stained hair follicles and/or the sweat gland duct, but not the epidermis. Antibodies, 34βB4, AE3, 34βE12, LP34, RCK102, MNF116, AE1, KL1, DE-K10 and DE-K13 reacted with every layer of the epidermis, hair follicles and the sweat gland duct. These results were similar to those reported in the human skin. No positive staining, however, could be detected in the epidermis, hair follicles and the sweat gland duct with commercial antibodies, 6B10, Ks.7.18, Mu146-uc, E3, RCK108 and involucrin. Therefore, immunohistochemical investigation with these commercial antibodies developed for human skin examination might be available for investigating the origin of skin tumors in dogs.

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A cat showing seasonal allergic symptoms of rhinitis was examined for reactivities to Japanese cedar (Cryptomeria japonica, CJ) pollen allergen by intradermal skin test (IDST), Prausnitz-Küstner (P-K) test, and lymphocyte blastogenic response. In IDST for 26 common allergens, the cat showed a positive reaction to CJ pollen allergen. P-K test using CJ pollen allergen also showed a positive reaction, indicating the presence of serum IgE specific to CJ pollen. In the lymphocyte blastogenic response, the stimulation index in the presence of CJ pollen allergen was 2.4. These data suggested that the seasonal rhinitis observed in the cat was caused by the sensitization to CJ pollen allergen.

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The testicular localization and expression of Smad2 and Smad3 mRNA involved in the intracellular signal transduction of activin, inhibin and transforming growth factor-beta (TGF-β) were examined under the influence of long and short photoperiod in Syrian hamsters (Mesocricetus auratus). In situ hybridization detected both Smad2 and Smad3 mRNA in spermatogonia and premeiotic spermatocytes in the active testis exposed to a long photoperiod, as well as in the regressed testis exposed to a short photoperiod. Northern blots showed that Smad2 mRNA was expressed at all stages over long and short photoperiods, whereas Smad3 mRNA was expressed at high levels in the photoperiod-induced regressed testis. The photoperiodic condition would change the balance between Smad2 and Smad3 transcripts in the testis. Thus, intracellular Smad2 and Smad3 might participate in transducing signals from activin, inhibin and TGF-β in spermatogenetic cells.

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A 20-year-old female Japanese macaque, weighing 8.7 kg, developed severe pulmonary acariasis. Numerous whitish nodules, 2-4 mm, were scattered throughout the lungs. Histologically multifocal granulomatous lesions consisting of a large number of eosinophils, epithelioid cells, foreign body type giant cells, and collagen fibers were aggregated around the mait bodies. Numerous mast cells were also detected in the lesions by toluidine blue staining, and tested positive for tryptase by immunohistochemistry. This may be the first reported case of severe pulmonary acariasis in a Japanese macaque.

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Esophageal carcinoma was observed in an eight-year-old, castrated male, Japanese domestic cat. Histologically, this neoplasm consisted of two different growth patterns, squamous cell carcinoma and adenocarcinoma. The results of immunohistochemical examination supported the fact that the two kinds of neoplastic cells have different characteristics. The tumor was, therefore, diagnosed as adenosquamous carcinoma. Esophageal tumors in the cat are very rare and, if any, neither adenocarcinoma nor adenosquamous carcinoma has been reported up to the present.

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A 2-year and 6 month-old, female, Golden Retriever showed circling behavior and seizure. By magnetic resonance imaging (MRI) examination, a mass was found on the surface of the left cerebral hemisphere, invading to the left temporal muscle. The skull bone between them was destroyed. The dog was euthanized and necropsied. Histologically, the mass contained a lot of undifferentiated anaplastic cells, forming Homer-Wright rosettes and pseudopalisading patterns. Thus, the case was diagnosed as primitive neuroectodermal tumor (PNET).

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In a 5-year-old Holstein cow, a neoplasm composed of a large intramuscular mass and multiple metastases in the lungs and lymph nodes was diagnosed as a pleomorphic rhabdomyosarcoma. This neoplasm was characterized by marked variation in tumor cell size and giant cells with single bizarre nuclei. Although the presence of cross striations and myoglobin could be confirmed, expression of alpha-smooth muscle actin (SMA) was also recognized in a few cells. Neoplastic cells showing intense staining for desmin, vimentin and proliferating cell nuclear antigen irrespective of their size differed from those in an embryonal rhabdomyosarcoma that exhibited a wide spectrum of differentiation, reminiscent of normal skeletal myogenesis. The cellular pleomorphism and SMA expression seemed to be characteristic of deviation from normal muscle cells or satellite cells in adult muscle.

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The effects of K⁺ channel blockers and P_2Y receptor agonist/antagonist on the vasorelaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) were investigated in the rabbit renal artery. Acetylcholine (ACh, 1 nM-10 μM) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 μM) in a concentration-dependent manner. N^G-nitro-L-arginine (L-NAME, 0.1 mM), an inhibitor of NO synthase, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was only partially inhibited by L-NAME whereas combined addition of L-NAME and 30 mM KCl completely inhibited the relaxation. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.1 μM) and apamin (1 μM), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P_2Y receptor antagonist, cibacron blue (10 and 100 μM) in a concentration-dependent manner. Furthermore, ADP_βS, a potent P_2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue alone. In conclusion, ACh may activate the release of ATP from endothelial cells which in turn activates a P_2Y receptor on the endothelial cells followed by a release of EDHF, resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K⁺ channels and the Ca²⁺-activated K⁺ channels.

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To characterize the mechanisms of acetylcholine (ACh)-induced vasorelaxation in rabbit renal arteries precontracted with high K⁺ (100 mM), muscle tension and cytosolic free Ca ²⁺ concentration ([Ca²⁺]_i) were measured simultaneously in the fura-2-loaded arterial strips. In the artery with endothelium, high K⁺ increased both [Ca²⁺]_i and muscle tension. Addition of ACh (10 μM) during high-K⁺ induced contraction significantly relaxed the muscle and induced additional increase in [Ca²⁺]_i. In the presence of NG-nitro-L-arginine (L-NAME, 0.1 mM), ACh increased [Ca²⁺]_i without relaxing the muscle. In the artery without endothelium, high K⁺ increased both [Ca²⁺]_i and muscle tension although ACh was ineffective, suggesting that ACh acts selectively on endothelium to increase [Ca²⁺]_i. 4-DAMP (10 nM) or atropine (0.1 μM) abolished the ACh-induced increase in [Ca²⁺]_i and relaxation. However, pirenzepine (0.1 μM), AF-DX 116 (1 μM) and tropicamide (1 μM) were ineffective. The ACh-induced increase of [Ca²⁺]_i and vasorelaxation was significantly reduced by 3 μM gadolinium, 10 μM lanthanum or 10 μM SKF 96365. These results suggest that, in rabbit renal artery, ACh-evoked relaxation of 100 mM K⁺-induced contractions is mediated by the release of endothelial NO. ACh may stimulates the M₃ subtype of muscarinic receptor in the endothelial cells, resulting in the opening of the nonselective cation channels followed by an increase of [Ca²⁺]_i and stimulation of NO synthase.

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The usefulness of magnetic resonance (MR) is already established, but it has a disadvantage of requiring a long scanning time. A short-time examination is more or less needed so as to be more practical in veterinary clinics. A protocol of the short-time MR examination was devised based on parameters determined, and validity of the protocol was assessed through the diagnosis of clinical cases with intervertebral disc diseases. With this protocol, it was possible to complete an MR examination for the spine within 15 min. The MR images and myelographic findings were correlated well in this study, suggesting the short-time protocol of MR examination can be used in the clinical diagnosis of spinal diseases.

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Two new canine melanoma cell lines (CMM1 and CMM2) were established from the patients with oral malignant melanomas. Histopathological type of both CMM1 and CMM2 was a mixed cell type consisted of spindle-shaped cells, polygonal cells, and oval cells. Doubling time of CMM1 and CMM2 were 18.4 ± 1.96 hr and 21.0 ± 0.73 hr, respectively. The effect of two kinds of retinoids (all-trans retinoic acid and 9-cis retinoic acid) on the proliferation of these cells were examined by morphological changes, proliferation assay and apoptosis assay. However, the retinoids did not suppress growth rate of these cells. This result suggests that retinoids used in this study did not induce differentiation, apoptosis, and growth inhibition of the canine melanoma cell lines.

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To elucidate the effects of ultrasound-guided transvaginal follicular aspiration, plasma concentrations of FSH, LH, inhibin, estradiol-17β and progesterone, and folliculogenesis were examined in Holstein cows. Four clinically healthy cows with regular estrous cycles were scanned by ultrasound per rectum once a week for 9 weeks before the commencement of follicular aspiration. All visible follicles were divided into 3 categories based on their sizes (2 ≤ small < 5 mm; 5 ≤ medium < 10 mm, large ≥ 10 mm). The follicular aspiration was started at random during the estrous cycle and conducted under epidural anesthesia induced with 5 ml of 2% lidocaine once a week for 6 weeks. The average number of total visible follicles ≥ 2 mm in diameter at 7 days after aspiration (21.7 ± 7.4, n=24) was similar to that before starting aspiration (26.7 ± 10.5, n=36). Plasma inhibin and estradiol-17β declined and fell to a trough on 1.5 days and returned to pre-aspiration values by 5 days after aspiration. Plasma concentrations of FSH increased and reached peak levels between 1 and 1.5 days after aspirations. Plasma concentrations of LH also increased and reached peak levels between 0.5 and 1.5 days after aspirations. Both plasma FSH and LH had returned to pre-aspiration levels by 5 days after aspirations. Plasma concentrations of progesterone did not change with the follicular aspiration. These results demonstrate that follicular aspiration decreases plasma concentrations of inhibin and estradiol-17β, which in turn leads to a rise in plasma concentrations of FSH and LH. It is suggested that marked increases in plasma concentrations of FSH and LH after the aspiration stimulate the development and maturation of a new cohort of follicles within one week in cows.

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To investigate the effects of intermittent and continuous exposure of lactating rats to Aroclor 1242 (a PCB congener), testis weight, daily sperm production (DSP) and Sertoli cell number per testis were examined in the adult male offspring. Thyroxine (T₄) was also measured because of the well-documented effects of polychlorinated biphenyls (PCBs) on this hormone. In experiment I, 3 groups of lactating female rats received daily subcutaneous injections of low (0.8 mg) or high (1.6 mg) doses of Aroclor 1242 in 0.1 ml corn oil from parturition to weaning of pups at 21 days. In experiment II, 3 groups of lactating rats received 2 subcutaneous injections per week of 0.8 or 1.6 mg Aroclor 1242, as in experiment I. In both experiments, control rats received vehicle alone. Serum T₄ was measured at 21 and 90 days of age, and testis weight, DSP and Sertoli cell numbers were examined at 90 days. In experiment I (continuous exposure), both the low (0.8 mg) and high (1.6 mg) doses suppressed T₄ concentrations at 21 days of age. Testis weight was increased by 14.8% (LD) and 16.5% (HD) compared with controls. DSP was increased by 20.4% in the low dose and 25% in the high dose animals compared with controls. The number of Sertoli cells per testis was increased by 32.6 and 39.4% in low and high dose animals, respectively. A similar study in which the lactating females were only dosed twice per week (experiment II) did not show any differences in these parameters. These results indicate that continuous exposure of lactating female rats to PCBs increases testis weight, sperm production and Sertoli cell numbers in the adult male offspring.

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The complete nucleotide sequences of the genes encoding two of the major inner capsid proteins of Ibaraki virus (IBAV), belonging to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) were determined. The L3 RNA segment is 2768 nucleotides in length which encodes VP3 polypeptides of 899 amino acid residues (M.W. 103 kDa). The S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and encodes 349 amino acids (M.W. 38 kDa). These RNA segments had the characteristic consensus motifs of Orbivirus RNA segments in termini, namely 5'-GUUAAA... and ...ACUUAC-3'. The comparison of the IBAV L3 and S7 sequences with those of other two EHDV-2 isolates revealed the higher homologies of 93% and 92% against EHDV-2 Australia isolate (EHDV-2AUS) and lower homologies of 80% and 81% against EHDV-2 North America isolate, respectively. The phylogenetic analysis based on L3 and S7 genes also indicated close relationships between IBAV and EHDV-2AUS.

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