Most metabolic diseases in dairy cows occur during the peripartum period and are suggested to be derived from fatty liver initially developed during the nonlactating stage. Fatty liver is induced by hepatic uptake of nonesterified fatty acids that are released in excess by adipose tissues attributable to negative energy balance. The fatty accumulation leads to impairment of lipoprotein metabolism in the liver, and the impairment in turn influences other metabolic pathways in extrahepatic tissues such as the steroid hormone production by the corpus luteum. Detailed understanding of the impaired lipoprotein metabolism is crucial for elucidation of the mechanistic bases of the development of fatty liver and fatty liver-related peripartum diseases. This review summarizes results on evaluation of lipoprotein lipid and protein concentrations and enzyme activity in cows with fatty liver and those with ketosis, left displacement of the abomasum, milk fever, downer syndrome and retained placenta. Obtained data strongly suggest that decreases in serum concentrations of apolipoprotein B-100, apolipoprotein A-I and apolipoprotein C-III, a reduction in activity of lecithin:cholesterol acyltransferase and induction of haptoglobin and serum amyloid A are intimately related to the development of fatty liver and fatty liver-related diseases. Moreover, determination of the apolipoprotein concentrations and enzyme activity during the peripartum period is useful for early diagnoses of these diseases.

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To study luteal function in the late gestational period of Phocidae (seals), we analyzed the localization of steroidogenic enzymes (P450scc, 3βHSD and P450arom) and prolactin receptors in the corpora lutea of pregnant spotted seals (Larga seal; Phoca largha) immunohistochemically. P450scc, 3βHSD and prolactin receptors were present in all luteal cells of each corpus luteum, and most luteal cells were immunostained for P450arom. Although we analyzed only two specimens, P450scc, 3βHSD and prolactin receptors were negatively immunostained in the placentae. P450arom was present in the syncytiotrophoblast of placentae. These findings suggest that 1) the corpus luteum of the spotted seal synthesizes pregnenolone, progesterone and estrogen during late gestational period, 2) the placenta of this species do not possess the capacity to synthesize progesterone, and 3) like other terrestrial carnivores, this species requires prolactin to maintain the corpus luteum during pregnancy. These characteristics support the recent classification of family Phocidae in the order Carnivora, and suggest a relationship between prolactin and reproductive failure during the post-implantation period in pinnipeds.

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Nucleotide sequences of cytochrome b/tRNA/D-loop region on mitochondrial DNA of mustelids feces were compared to identify species. PCR amplification of target sequence for 47 (24.9%) feces and species identification of five feces (2.6%) out of 189 feces, collected at several study sites in Hokkaido, were successful. Species of three feces were Martes zibellina and those of other two feces were Martes melampus and Mustela itatsi. The low success rate of identification appeared to be due to failure of PCR amplification by inhibitors in feces. It was suggested that the method used in this study was useful for not only identify mustelids species, but also analyzing their genetic relationships.

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Leptin is a protein synthesized and secreted primarily by adipocytes, and the circulating leptin concentration is elevated in obese humans and rodents. Recently, we have established a sandwich enzyme-linked immunosorbent assay for canine leptin. In the present study, plasma leptin concentrations were measured in experimentally developed obese beagles and in clinically obese dogs. When 5 male beagles were given a high-energy diet for 3 months, all of them became obese and the plasma leptin concentration significantly increased from 2.4 ± 1.2 to 4.9 ± 0.9 ng/ml, positively correlating with body fat content estimated by the deuterium oxide dilution method (r=0.87). The leptin concentrations of plasma samples collected from 59 dogs in veterinary practices were compared with their body condition scores (BCS). The plasma leptin concentrations of obese dogs were 9.7 ± 0.7 and 12.3 ± 1.5 ng/ml at BCS=4 and BCS=5, respectively, which were significantly higher than those of optimal (BCS=3) dogs (2.7 ± 0.3 ng/m l). There was no significant effect of sex and breed. A weak positive correlation (r=0.37) was found between the plasma leptin concentration and age, probably due to the lesser content of visceral fat in puppies younger than 1 year old. These results indicate that plasma leptin is a good index of adiposity in dogs regardless of breed, age and sex, and may be useful for quantitative assessment of obesity in small animal practice.

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Loss of adenosine-5'-triphosphate (ATP) and accumulation of inosine-5'-monophosphate (IMP) are the major purine metabolic changes in the skeletal muscle during hypoxia. This study addressed whether chemical metabolic inhibition reflects those changes in cultured skeletal myotube. For this aim, mouse-derived C₂C ₁₂ myotubes were cultured in Hank's balanced saline solution containing 2 mM sodium cyanide (CN) and/or 1 mM iodoacetic acid (IAA) up to 180 min. Inhibition of oxidative phosphorylation by CN induced a minimal change in the intracellular adenine nucleotide levels during 180 min. Blockage of glycolysis with IAA caused an over 90% decrease in adenine nucleotides both in the cytoplasmic and intramitochondrial spaces, accompanied with allantoin release. Since 1 mM allopurinol entirely inhibited the allantoin generation, xanthine dehydrogenase/oxidase was found to play a key role in the purine catabolism in IAA-treated C₂C₁₂ myotubes. By the combined treatment with CN+IAA, ATP exhaustion and IMP accumulation was achieved with significant cell injury. These changes were comparable with those in skeletal muscles during hypoxia, indicating that our model with CN+IAA is well applicable to the investigation of hypoxia-induced myopathy.

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³H-thymidine (³H-TdR) incorporation assay has been generally used to measure lymphocyte proliferation in the chicken. Disadvantages of this assay are that radioisotope is biological hazard to the person and environment and that it can not measure which subset of lymphocytes proliferates. In this study, bromodeoxyuridine (BrdU) incorporation assay by flow cytometry was compared with ³H-TdR incorporation assay. As a result, BrdU incorporation assay showed a strong correlation with ³H-TdR incorporation assay, and it could be applied simultaneously to detect BrdU incorporation and expression of cell surface marker antigens. These results suggest that the BrdU incorporation assay by flow cytometry is useful to analyze lymphocyte proliferation in detail.

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Urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) was examined in healthy dogs and dogs with urinary diseases, and its clinical usefulness as an indicator of urinary diseases was discussed. Twenty-eight healthy dogs and 20 dogs with urinary diseases were used. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-β-D-glucosaminide as substrate, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG index in urine of healthy dogs was 3.2 ± 2.4 U/g, and NAG index in the dogs with chronic renal failure or lower urinary tract infection accompanied by pyelonephritis was higher than that in healthy dogs. However, the dogs with lower urinary tract infection without pyelonephritis showed normal values of NAG index. Some dogs with diabetic mellitus showed elevated values of NAG index when control of blood sugar was not successful. Increase of NAG index was observed in some dogs with pyometra before increases of BUN and serum creatinine concentration. Therefore, NAG index in urine seems to be a good indicator for urinary diseases in dogs.

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The present study was designed to assess the clinical usefulness of measurement of urinary N-acetyl-β-D-glucosaminidase (NAG) activity and its isoenzymes in cats with urinary disease. Thirty-five healthy cats and 9 cats with renal disease were used. Furthermore, a 5-year-old female cat was administered a large amount of sulfonamide in order to induce acute renal failure, and urine samples were collected for the assay of NAG activity and its isoenzymes. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-β-D-glucosaminide, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG isoenzymes were assayed by use of the mini-column method and electrophoresis. The overall mean value of urinary NAG index in healthy cats was 1.6 ± 1.5 U/g. Urinary NAG index varied from 6.2 to 35.5 U/g in cats with chronic renal failure. There was no significant correlation between BUN, serum creatinine concentration and urinary NAG index. In cats with feline lower urinary tract disease, normal values of urinary NAG index were observed. In the urine samples of healthy cats, the proportions of NAG isoenzyme A (NAG-A) and isoenzyme B (NAG-B) were 79.1 ± 4.4 % and 21.0 ± 4.4 %, respectively, as assayed by the mini-column method. In the assay of NAG isoenzymes by electrophoresis, the proportions of NAG-A and NAG-B in healthy cats were 66.6 ± 5.8 % and 33.4 ± 5.8 % , respectively. The proportion of NAG-B as measured by electrophoresis was significantly larger (p<0.05) than that obtained with the mini column method. A feline case of acute renal failure experimentally-induced by sulfonamide showed elevation of urinary NAG index, NAG-A and NAG-B after injection of sulfonamide. The increase in NAG-B was larger than that of NAG-A. From the results reported here, measurement of urinary NAG and its isoenzymes seems to yield information about tubular damage at an early stage in cats with urinary disease.

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It is difficult to produce homogeneous cell suspensions of Malassezia pachydermatis, since yeast cells paste up and form many clumps. However, homogeneous fungal suspensions are required for susceptibility examinations and biochemical analyses. Although several types of trials have been carried out using glass homogenizers and many types of agents to obtain homogeneous fungal suspension. They have not yielded good results. We therefore attempted to use an ultrasonic homogenizer to separate clumps of yeast cells into separate individual cells. We succeeded in this fashion in producing homogeneous cell suspensions of M. pachydermatis. These results indicate that an ultrasonic homogenizer can be used to prepare homogeneous fungal suspensions of M. pachydermatis.

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Although heart failure in cats is treated with angiotensin converting enzyme (ACE) inhibitors, data on the effects of different doses of enalapril on hemodynamics and the inhibition of ACE activity have not been published. To evaluate the effect of enalapril, 0.25, 0.5, or 1.0 mg/kg was given once (s.i.d., p.o.) or twice (b.i.d., p.o.) a day, and plasma ACE activity, indirect blood pressure, and heart rate were measured. Plasma ACE activity and blood pressure fell dose-dependently. There was a biphasic effect on blood pressure with twice daily administration. Enalapril 0.25 mg/kg b.i.d. inhibited plasma ACE activity by 40% after 24 hr, which was almost the same as the effect of 0.5 and 1.0 mg/kg s.i.d., and 0.5 and 1.0 mg/kg b.i.d., while 0.25 mg/kg s.i.d. inhibited it by 23%. Thus, enalapril with a daily dose exceeding 0.5 mg/kg may provide similar efficacy of ACE inhibition in cats.

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Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/ 2,019) and 2.2% (44/ 2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/ 109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them was seropositive for both infections. Based on the results of this study, further investigations should be required to survey horses that have arrived in Japan relatively recently and tick vectors of equine Babesia using ELISA with some recombinant protein, a parasite detection method in an in vitro culture of equine Babesia, and PCR testing.

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Using macrophage scavenger receptor-A knockout (SRKO) mice, we examined the role of macrophage class A scavenger receptors (MRS-A) on the immune response and acquisition of host resistance against repeated infestation with Haemaphysalis longicornis. Except for one batch of nymphs that infested one of the SRKO (SR-/-) mice and showed no appreciable reduction in body weight, all the other groups of nymphs manifested significant decrease in body weight. Both SR-/- and wild type (SR+/+) mice showed a sustained increase in anti-tick antibody titers, but SR+/+ mice showed significantly higher titers. The IFN-γ assayed in SR-/- mouse immune sera was substantially less compared with that in SR+/+ mice. Immune sera from SR-/- and SR+/+ mice recognized the 51 and 44 kDa, and 44 kDa proteins, respectively, of the salivary gland antigen. The difference in the level of anti-tick resistance manifested by both groups of mice may be influenced by less efficient trapping and processing of tick antigens by macrophages in mice lacking for the macrophage scavenger receptors, and consequently affected the cascade of Th1 and Th2 responses. We have thus obtained valuable data that strongly infer the role of MSR-A in enhancing host defense against repeated infestation with H. longicornis.

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A case of olfactory neuroepithelioma was investigated electron microscopically and immunohistochemically. The tumor mass was found in the nasal cavities of a 10-year-old female dog, which showed epistaxis, nasal discharge and facial swelling. The tumor tissue consisted of tubular structure of cuboidal to columnar cells and compactly arranged nests of small cells surrounded by a fibrovascular stroma. Mitotic figures were frequently observed. Immunohistochemically, the tumor cells frequently showed positive for neurofilament protein, synaptophysin and/or carnosine in addition to keratin. Ultrastructurally, tight junction was observed between the tumor cells. No dense-cored secretory granules were shown in the tumor cells. These findings indicated that the present tumor had neuronal and epithelial features probably originating from the olfactory epithelium.

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To determine the effects of rapid infusion of essential fluids in a volume of hypotonic lactated Ringer's solution, the central venous pressure (CVP) and acid-base equilibrium were investigated in to mildly dehydrated heifers. Mild dehydration was induced in 9 Holstein heifers by withholding food and water until 7.0 ± 5.7% of plasma volume had been lost. The heifers were randomly assigned to the ILG (lactated Ringer's + 5% dextrose), HLG (1/2 lactated Ringer's + 2.2% dextrose) or HRG (1/2 Ringer's + 2.5% dextrose) groups with 3 heifers in each group. Heifers received 30 ml/kg of one of the fluids, at a flow rate of 20 ml/kg/hr. The rapid intravenous (IV) infusions of HLG and HRG used in this study were found to be safe and effective in increasing plasma volume without increasing CVP, even though the infusion was given to the jugular vein at a dosage of 30 ml/kg. However, ILG infusion induced progressive increases in CVP, reaching 9.0 ± 2.0 mmHg. No clinical signs, such as moist rales on auscultation, moist cough, jugular vein congestion, ophthalmoptosis, salivation or arrhythmia, were observed throughout the fluid infusion. The relative changes in base excess (rBE) for the ILG and HRG groups were significantly decreased until the end of fluid infusion. As for the HLG group, rBE slightly decreased until the end of the fluid infusion. Then the values significantly increased and exceeded the pre-infusion value at the end of the experiment. While IV infusion of HLG inhibited acidification caused by dilution, HRG infusion induced diluted acidification. It is suggested that HLG infusion should be examined as a treatment for cattle with dehydration and moderate metabolic acidosis, since rapid infusion of HLG may be more beneficial for rehydrating cattle with metabolic acidosis than current treatment.

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Fecal estradiol concentrations were measured in three captive unmated female sun bears (Helarctos malayanus) from August 1998 to July 1999 in Sarawak, Malaysia and vaginal smears from one of the females was observed in August 1998 and March 1999. A single peak in fecal estradiol concentration was obvious for each bear in August or September 1998, and there was a much higher percentage of superficial vaginal anuclear cells in August 1998 than in March 1999. These results suggest that sun bears in Sarawak are likely to be a seasonal breeder associated with a peak of estrogen production in August or September.

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Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4°C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean ± SD) of motile and live sperm were 96 ± 2 and 86.5 ± 7.2% before freezing, and 43 ± 5 and 67.4 ± 3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8 ± 1.2 × 10⁸ cells/ejaculate), frozen semen in the present study might serve for artificial insemination.

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Two inbred strains of Hatano rats, which had been bred selectively on the basis for shuttlebox avoidance responses (high- and low-avoidance animals; HAA and LAA), were different in sperm motility. We have found that there are strain differences in the spontaneous incidence of sperm morphological abnormalities. The percentages of abnormalities were extraordinarily higher in the LAA rats, whose sperm motility was lesser. Since the high sperm abnormality was an exceptional case in rats, these rats may be useful models for sperm quality.

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The age-related changes of vaginal opening, body weight, the weights of the uterus and ovary, together with histological examination, serum 17β-estradiol (E2) and progesterone levels were examined in intact female Crj:CD^® (SD) IGS rats between 21 and 36 days of age to understand the basic biological profile of changes of the female genital system during sexual maturation in the rat for female pubertal assays. With the beginning of the elevation of serum E2 level from 28 days of age, all parameters except body weight started to show drastic change until 31 days of age. The highest incidence of vaginal opening was recorded at 34 days of age. On macroscopic examinations, a number of rats showed uterine imbibition but vaginal opening. Immediately after the confirmation of the vaginal opening, the genital systems of three rats were observed microscopically. Both ovaries already had multiple corpora lutea, and degeneration of endometrial epithelial cells was observed. In conclusion, we obtained essential data on genital tract development of female Crj:CD^® (SD) IGS rats for in vivo screening assays that will contribute to detect potential endocrine active chemicals. In addition, it is assumed that the first ovulation precedes or occurs simultaneously with vaginal opening.

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