1. Bovine a1-acid glycoprotein (α1-AG) was purified from polled normal bovine sera by successive ammonium sulfate precipitation, ion-exchange chromatographies and gelfiltration. Bovine α1-AG had a molecular weight of 42, 000±2, 000 and sedimentation coefficient of 3.4s. It contained 26.6% carbohydrate. Gel isoelectric focusing revealed a microheterogeneity with 7 to 8 bands in a pI range of 3.2 to 3.7. It migrated to the α1-globulin region upon immunoelectrophoresis. Single radial immunodiffusion was developed for the quantitative measurement of bovine α1-AG in serum.
2. Serum α1-AG levels increased during fetal development and neonatal stage. Mean ± SD serum α1-AG concentration reached a peak of 1, 368±207μg/ml immediately after birth, but thereafter gradually decreased, to 249±100, μg/ml, similar to the normal adult bovine range.
3. By use of isoelectric focusing of thin-layer gels, detected 7 microheterogeneity bands ranging from pI 3.2 to 3.8 in adult bovine serum. Twelve bands ranging from pI 2.6 to 3.8 were found in the 9-month fetus and in neonates. The 5 most acidic bands, which are absent in adult serum, ranged between pI 2.6 and 3.1 and decreased with maturation as band patterns assumed adult characteristics. By crossed affinity electrophoresis, α1-AG of adult bovine serum was separated into 4 peaks according to its differential affinity to concanavalin A (Con A). Seventy-five percent of the α1-AG concentration was represented by peak 3 (P-3) and peak 4 (P-4), which had moderate or strong binding to Con A. In contrast, fetal sera contained only peak 1 (P-1) which did not have Con Abinding affinity. In neonatal sera, 4 peaks were recognized, of which P-1 comprised 70% of the total α1-AG present. Thereafter, with aging, percentage of P-3 and P-4 increased as band composition approached the normally expected adult pattern.
4. Single radial immunodiffusion was developed to the guantitative measurement of bovine α1-AG in serum. The mean serum values of α1-AG in 152 healthy Holstein cattle (1-12 years old) was 283.2±82.3μg/ml. Elevated values (cut-off value=450, μg/ml) were observed in cattle with liver disorders (92%), pneumonia (92%), traumatic pericarditis (89%), peritonitis (100%), perinatal unability to stand (93%), fracture dislocation (100%), jones disease (100%), bovine viral diarrhea (100%), bovine leukemia (79%), and metabolic disorders (17%). Cattle with inflammatory diseases showed remarkably high α1-AG levels (1, 116.7±858.7μg/ml). In group I (recovered cases) showed almost the same level of serum α1 -AG abnormal cattle through the investigation period. The high levels returned to the normal levels according to the improvement of cilinical symptoms. In group II (unrecovered cases) maintained high levels of about over 1, 000μg/ml during the observation period. These animals were slaughtered or autopsied within a short period after initial examination.
5. In the cases with laparotomy, the levels of almost recoverered cattle reached a maximum 3-7 days after operation, and thereafter recovered to the preoperative or normal level 15-40 days after operation. In the case with postoperative peritonitis, the high serum α1-AG level was maintained high until autopsy. On the day before autopsy, the level was further increased. Those results suggest that serum α1-AG level in recovered cases was not incresed, while it was high in unrecovered cases. As an indicators for the diagnosis of inflammatory disease, the assay of serum α1-AG seemed to be very useful for evaluation of an appropriate treatment and of condition of postoperative course in cattle.

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Radiographic and morphological investigation was performed to establish the accurte antemortem diagnostic technique for wobbling foals. 1) There were radiographic abnormalities on cervical radiography in 17 out of 19 wobbling foals. Twelve out of those 17 foals had severe histopathological lesions in the white matter of the spinal cord at the level of the vertebrae with radiographic abnormalities. Thus, radiographic examinations including myelography on cervical vertebrae is a quite important and effective diagnostic aid for wobbling foals. 2) As a result of discriminant analysis on cervical vertebrae of the foals with the age of 13 months or elder consisting of 28 ataxic foals and 19 control foals, sufficient discrimination was performed with the high hit rates on C3-C7. 3) Ratios of stenosis of spinal canal on myelography and survey radiography were defined on radiograms of lateral flexed position of mid-cervical region in 12 wobbling foals. When the standard value of the ratio of stenosis on myelography was set at 40%, the stenosis and histopathological lesions at the same site was well coincided in 10 out of 12 foals. When the standard value of the ratio of stenosis on survey radiography was set at 40%, the stenosis and histopathological lesions at the same site was well coincided in 9 out of 12 foals.

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Six newborn Japanese Black calves with severe hemolytic anemia were noted from 1991 to 1993. All calves showed typical spherocytosis in which most of the cells were microcytic spherocytes deprived of the central pallor, and it was also clear that osmotic fragility of their red cells was significantly increased. Autoimmune disorder, enzymopathies disorder. red cell membrane protein were examined, to investigate abnormalities of Red cells. In consequence it was made clear that 6 cases were "Red blood cell membrane protein abnormality" with band-3 deficiency. It was clear that, hereditary spherocytosis in human was occurred in autosomal dominant transmission. Red cell membrane protein deficiency in Japanese black cattle. To reveal that these cases were occurred in hereditary, clinical test, blood and biochemical examinations, shape of red cells, osmotic fragility test and analysis of membrane proteins were performed in 6 dams and 15 non-consanguinty calves. There was significantly higher number of spherocyte in the 6 dams as compared to the non-consanguinty calves. There was significantly mild increased osmotic fragility in the 6 dams compared to the non-consanguinty calves. Osmotic fragility of dams were significantly higher than that of non-cosanguinty normal cattle in a rate of hemolysis in 0.5% NaCl at fresh and 24 hour incubated blood (p<0.01). Particularly, to compare the existent ratio of band-3 and 4.2 in each groups, existent ratio of band-3 and 4.2 in dams showed 63%, 80% respectiely. It was made clear that those of dams developed middle value between affected calves and non-consanguinty calves. Based on their findings, mode of hereditary was examined. It 6 affected calves (band-3 deficiency) were Homotype (AA), dams and a part of siblings (middle values of band-3) were Hetrotype (Aa), non-consanguinty calves (complete developement of band-3) were non-carier type (aa), these cases were determined as "Autosomal incomplete dominant transmission" in all cases. According to assum genotype used by osmotic fragility test, cows for production in A fame were diagnosed genotype and all cows were mated with bulls assumed non-carrier after control. The effect of these control were observed, according with comparison of genotype of newborn calves during pre-control (1991-1992) and post-control (1993-1994). Frequency of genotype changed form 54% to 22% before and after control. When genotype of cows and bulls were diagnosed respectively, possiblity of hereditary control was proved in clinical field.

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The following studies were carried out to clarify the relationship between the outbreak of hyena disease and the administration of vitamin AD3E (V-A D3E) premix. (1) In the examination of clinical cases, presence and dosage of V-AD3E premix were surveyed in 11 cattle aged 12 to 17 months and diagnosed as hyena disease. As the results, all of them were found to be administered excessive amount of V-AD3E premix within 1 month after birth. The bone morphometric analysis in 2 cases showed the decreased bone formation due to low turnover in bone metabolism. (2) Based on the results of clinical studies, the excessive amount of V-AD3E premix, vitamin A (V-A), or vitamin D3 (V-D3) was experimentally administered in calves to cause the outbreak of hyena disease. Each vitamin compound was daily administered to calves aged 7 days after birth for 10 days. The hyena disease was recognized in 2 out of 2 calves administered a high dose of V-AD3E premix (V-A 3, 000, 000, V-D3 300, 000, and V-E 1, 200 LU./day), 1 out of 2 calves administered a half dose of the V-AD3E premix as above described, and 1 out of 2 calves administered with only V-A (V-A 3, 000, 000 I. U./day). Two calves administered only V-D3 (V-D3 300, 000 I. U./day) did not develop the hyena disease. In all hyena disease calves, the plasma retinylpalmitate level remained high until 1 month after birth. The bone metabolism was in the state of low turnover at 50 days after birth and the bone volume was small at 12 months after birth. All hyena calves showed the flat chondrocytes, the decrease in chondrocytes, and the irregular structure of column in the epiphysial growth plate. (1) In conclusion, the excessive administration of V-A to calves resulted in the hyena disease, which attributed the cause of this disease to V-A.

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