Antibody titers against canine parvovirus (CPV), canine distemper virus (CDV) and infectious canine hepatitis virus (ICHV) in serum were measured in 6 beagle dams and their 38 puppies bred in our colony, in order to clarify the effects of maternally derived antibodies to antibody responses against the viruses after vaccinations in puppies. Correlation coefficient on antibody titers between puppies and dams were CPV: r=0.7935, CDV: r=0.8194 and ICHV: r=0.8105. Mean maternal antibody positive rates in 7-day-old puppies from their dams were CPV: 67%, CDV: 46% and ICHV : 45%. Mean half-lifes of the maternal antibodies in puppies were estimated to be CPV: 13.5 days, CDV: 15.1 days and ICHV: 15.4 days. The antibody response against CPV vaccination in puppies was mainly observed in dogs being titers of less 1: 5 and positivity was 39% (15/38 puppies) after 1st vaccination at 42 days after birth, and 82% (31/38 puppies) after 2 nd vaccination at 70 days. That against CDVvaccination (at 56 days after birth) was seen highly in dogs being titers of less 1: 10 and positivity was 53% (20/38) . Also that against ICHV vaccination (at 56 days after birth) was seen frequently in dogs being titers of less 20 holds and the rate was 87% (33/38) . From the these results, it was estimated that the age when high antibody response against each vaccination could be expected in puppies might be CPV: between 40 and 69 days, CDV: between 32 and 92 days and ICHV: between 31 and 52 days, respectively.

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The influence of long term rearing conditions on the hematological and serum biochemical values in laboratory-bred female cynomolgus monkeys (Macaca fascicularis) was studied under the following two conditions. Group A: New born infants were nursed by their mothers for at least 12 weeks after birth. After weaning, four monkeys of approximately equal age and body weight were kept in a relatively small cage (90cm W×60cm D×60cm H) and reared to five years of age. These rearing conditions are commonly used in the breeding program of our primate center. Group B: One male and five females of wild origin were introduced into a relatively large cage (180 cm W×180 cm D×160 cm H) . Female monkeys born in these cages were used in this study. All of them were reared by their own mothers and grew well in these cages. Male monkeys born in these cages were removed at about 3.5 years of age. Hematological and serum biochemical values between the two groups in each age class were compared and analyzed by the canonical discriminant analysis (the discriminant analysis with reduction of dimensionality) . Age correlated highly with the value of the first canonical variate. The second canonical variate detected a difference in the blood hematology and biochemistry probably induced by the difference in rearing conditions.

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Eight male DBA/1J mice immunized twice by intradermal injection of type II collagen were autopsied 12 weeks after the first immunization and analyzed for anti-type II collagen antibody level, and the limb joints were examined radiologically and histopathologically. Clinical onset of swelling and erythema in the limb joints occured about 5 weeks after the first immunization and deformity of the limbs was observed in a few animals about 5 weeks later. Although there were marked individual differences, serum anti-type II collagen antibody levels were elevated in all animals. Histopathologically, the changes were similar to those seen in human rheumatoid arthritis and were characterized by proliferation of synovial lining cells, formation of granulation tissue with destruction of cartilage and subchondral bone, and ankylosis. Systematic examination of various joints of the fore-and hind-limbs revealed definitely that the sequence of arthritic lesions was not uniform. The knee joint was involved most frequently, but smaller joints such as the phalangeal joints were involved less frequently but exhibited severe changes. The significance of histopathological examinations in the evaluation of effects of anti-rheumatic drugs was discussed with reference to this model.

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Experimental gastric ulcer formation was performed in the pika and compared with that in the rat. Gastric ulcers were formed in pika that were subjected to water restraint for 4-5 days for 2 hours each day. Gastric ulcers were also formed under conditions of 1-4 days for 3 hours each day and 1-2 days for 5 hours each day. The severest (widest) ulcers were obtained under the condition of 5 hours' water restraint. Histopathologically, the ulcers were mostly erosions, but those formed under 5 hours' restraint reached the tunica muscularis mucosae. In addition, inflammatory changes were recognized. In contrast, while gastric ulcers in the rat formed within a short time, they were histopathologically less severe than those in the pika. Therefore, water restraint for 4 hours performed 4-5 times is suitable to obtain gastric ulcer formation in the pika and may result in more severe gastric ulcers than in the rat. Compared with the rat, the pika showed differences in the appearence and degree of gastric ulcers formed by the injection of serotonin and reserpine.

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We biometrically analyzed the body weight growth data of new-born squirrel monkeys, obtained during the nursing period from 0 to 12 weeks of age. Body weight (y in grams) could be expressed as a function of birth weight (a in grams) and age (x in weeks) by the following equation: y= a + b x, where b indicates growth rate. This equation corresponded significantly with actual growth curves (R²=0.96) . The frequency distribution of b values was demonstrated to be anormal distribution. This value was used to judge whether the body weight growt hof each monkey was normal or abnormal. The lower control limit (LCL) wascalc ulated by using a linear equation with the b value of 9.07 (M-1.25×S. D.) and each birth weight. For the monkeys whose body weight was above the LCL during the first three weeks after birth, it was determined whether the frequency of weighings could be reduced from 13 to 7. Using the same animals, no significant difference was detected between the b value estimated from 13 measurements and that estimated from 7 measurements. Thus, from the standpoint of management's policy to save labor, the frequency of weighings could be reduced. A new daily routine has been established in our primate center to save labor by reducing the number of body weighings of the many infant monkeys. In the new program, newborn monkeys whose body weight is above the LCL are weighed only 7 times during the nursing period of 12 weeks, while those whose weight is below the LCL are weighed 8 to 13 times.

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The bronchial sensitivity to acetylcholine (ACh) of guinea pigs of various strains was investigated to clarify strain differences. Inbred Strain 2, Strain 13 and JY-1 and non-inbred Hartley strain (two colonies) were used in this experiment. (1) Guinea pigs were exposed to 0.08% ACh aerosol and the time needed to produce falling down (TNPFD) was determined. Mean ± standard error of TNPFD (n= 14 per group) of animals was 182±28 sec, 148±22 sec, 210±30 sec, 342±24 sec and 406±36 sec in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. There was a significant difference in TNPFD between inbred strains and non-inbred strains (P <0.05 or P <0.01), indicating that inbred strains had higher sensitivity. (2) Guinea pigs were exposed to 20-5000 pg/ml ACh for 2 min. The mean dose threshold as determined by transcutaneous oxygen pressure was 524μg/ml, 424μg/ml, 614pμg/ml, 1317μg/ml and 1651 μg/ml (n =14 per group) in Strain 2, Strain 13, JY-1, Hartley (Japan SLC) and Hartley (Hitachi), respectively. Inbred strains showed lower dose thresholds than non-inbred strains. (3) Isolated trachea-lungs of 5 guinea pigs were perfused with 10^-9-10^-5g/ml ACh to determine strain differences. Dose response curves of animals of inbred strains shifted to the left (lower concentrations), unlike those of non-inbred strains, suggesting that inbred strains had higher sensitivity to ACh than non-inbred strains. These results suggested that bronchial sensitivity was markedly higher in inbred strains than in non-inbred strains, whereas the differences between the three inbred strains and the two non-inbred colonies were not significant.

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The mandibular glands of Dasyuroides byrnei were examined by light microscopy, and transmission and scanning electron microscopy. The secretory units consisted of numerous seromucous acini and a few seromucous demilunes. The seromucous acini were almost always capped by demilunes. The acinar seromucous cells contained faintly basophilic, light, coarse, bipatite secretory granules with matrix of low and moderate densities. The demilunar cells were dark compared with acinar seromucous cells and contained acidophilic secretory granules with a fibrillogranular matrix of moderate density. Preacinar cells with a seromucous nature were occasionally present at the junction between the acinus and intercalated duct. These cells had numerous basophilic granules, which were similar to those of acinar seromucous cells. The intercalated ducts consisted of simple cuboidal light cells that had a few small electron-dense granules. The striated ducts were composed of tall columnar light cells containing numerous vesicles, but no secretory granules. The mandibular acini of D. byrnei were composed of two cell types having a seromucous nature, unlike those of the opossum and many other mammals.

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In a previous study, the author found that serum testosterone (T) levels in male beagle dogs showed a circadian rhythm which was lowest at 12: 00, and increased to a peak at 18: 00-6: 00, thereafter decreasing until 12: 00. The reason was thought to be that dogs were breeding under rigid controlled conditons. The present study was performed to investigate the effects of lighting on the circadian rhythm of T level by means of a reversal of the lighting time zone, because lighting is considered an important factor in modulating T levels. Six male beagle dogs of 2 years were used in this study. The routine lighting time (8: 00-20: 00) and dark time (20: 00-8: 00) zone in the breeding room was reversed completely and T levels were measured at intervals of 1-5 weeks for up to 54 weeks. Blood samples were collected at 6: 00, 12: 00, 18: 00 and 24: 00. The results showed that the circadian rhythm of the T level and the T level at each blood sampling time did not change significantly within 54 weeks. As a result, it was recognized that the circadian rhythm of the T level in male dogs may not be affected by changes in the lighting time zone.

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Postnatal behavioral development and learning ability of operant performance were examined in spontaneously epileptic rats (SER: zi/zi, tm/tm), and the original tremorous mutant strains of rats, tremor rats (tm/tm) and zitter rats (zi/zi) and their controls. Before the eyes opened, the increase in body weight and the age of achieving the righting reflex on a surface were not significantly different between the SER and their littermates without epileptic seizures (SER-N: zi/zi, tm/+ orzi/zi, +/+), and between tremor rats and the original strain Kyo: Wistar rats. After the eyes opened, the increase in body weight, age of achieving the righting reflex in air and traction performance, and the development of rotarod performance, were delayed in SER and tremor rats in comparison with other groups of rats. The zitter rats were apparently inferior in their development of rotarod performance in comparison with the same zitter homozygous SER-N. Operant performance was more inferior in SER than in SER-N and in tremor rats than in Kyo: Wistar rats. The differences were much more marked between SER and SER-N than between tremor and Kyo: Wistar rats. Thus, homologoustmgenes and the coexistence of homologoustmandzigenes have an inhibitory effect on postnatal behavioral development and learning ability.

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Effects of various types of motion stimuli were compared to investigate optimum method to elicit motion sickness and adaptation inSuncus murinus (suncus) . Three different direction of shaking in the horizontal plane, back and forth, right and left and revolving, induced emetic response to the similar extent. However, vertical shaking was far less effective in inducing motion sickness. Mild and severe horizontal shaking (15 min per day) was continued for 14 days and emetic response to standard motion stimulus was compared before and after the training. The severe daily acceleration strongly depressed the susceptibility to motion stimulus. The mild acceleration which was not emetic stimulus in itself also remarkably attenuated the vomiting response to standard motion stimulus. These results indicate that 1) the emetic responsiveness of the suncus does not depend on the modes of shaking as long as the direction is in the horizontal plane, 2) the suncus is relatively refractory to the vertical linear acceleration and 3) the adaptation to motion stimuls does not develop on the latest peripheral steps of the vomiting reflex pathways.

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To determine the chronic toxicity of diesel exhaust, F 344 rats were made to inhale various dilutions of exhaust under specific pathogen-free conditions for 16 hours a day, 6 days a week for 30 months. Five experimental groups of 240 rats each were set up, each consisting of equal numbers of rats of the both sexes of five weeks-old. During the experiment, they were housed in inhalation chambers, which were maintained at 23±2°C and 55±10% relative humidity with artificial lighting 12 hours a day. Daily water intakes of all rats in inhalation chambers during the period of flow of fresh air with lights on and the period of inhalation of exhaust with lights off were measured with a drip meter fitted to automatic watering system in each inhalation chamber throughout the experiment. Result was that the mean daily water intake of the 0.5mg/m³ (lowest particulate concentration level), 1.0mg/m³, 1.8mg/m³, and 3.7mg/m³ (highest particulate concentration level) groups were 15%, 24%, 27%, and 39% less than that of the control group. Moreover, the groups also showed dose-dependent decrease in hourly water intake during the period of inhalation of diesel exhaust with lights off and dose-dependent increase in that during the period of flow of fresh air with lights on. This finding suggested that diesel exhaust might affect the circadian rhythm of the water intake in rats. These results showed that the measurment of the water intake of rat is an useful parameter to detect the biological effect on inhalation experiment.

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The prototype of an ELISA kit using protein A as the second reaction reagent for mice and anti-rat IgG for rats was prepared for seromonitoring of the Sendai virus and mouse hepatitis virus (MHV) /sialodacryoadenitis virus (SDAV) /Parker's rat coronavirus (PCV) infections. The respective antigen strains and protein concentrations were Sendai virus MN strain, 2μg/ml and MHV Nu-67 strain, 5μg/ml. The reliability of this prototype kit was investigated in two field tests performed on a total of 10, 094 mouse and rat sera from 147 institutions. The results indicated that the two types of kits for the two species of animals were highly specific, but it is necessary to increase the detection sensitivity of the MHV antigen for the MHV antibody of mice and SDAV/PCV antibodies of rats.

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Improvement of the mouse hepatitis virus (MHV) antigen in a prototype ELISA kit was performed. Equivalent divalent antigens of MHV Nu-67 and S strains with a protein concentration of 10μg/ml showed the best sensitivity and specificity for the detection of MHV and sialodacryoadenitis/Parker's rat coronavirus antibodies in mice and rats, respectively. An increase in the reliability of macroscopic evaluation of both antibody tests is expected by using the newly manufacturedkit with the improved antigen.

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Detection of Mycoplasma pulmonis was examined by using the polymerase chain reaction (PCR) for amplifying a specific DNA sequence. In gel electrophoresis which was conducted to detect the amplified products, only 1 pg ofM. pulmonisDNA could be detected following 30 cycles of amplification, while no amplified product was detected even from 1 g ofM. arthritidisorM. neurolyticumDNA. Furthermore, 10 colony-forming units ofM. pulmoniscould be detected by direct amplification from the mycoplasma suspension. These results suggest the usefulness of the PCR as a highly sensitive, specific, and rapid method for direct detection ofM. pulmonis.

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The vomeronasal organ (VNO) of female Wistar-Imamichi rats was removed completely. The females were impregnated and gave birth to young. The question addressed was whether such dams would show deficits in nursing behavior. The data indicated that removal of the VNO severely depressed nursing behavior. We conclude that the VNO contributes to the nursing behavior of lactating rats.

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Conventional DBA/2 male mice of about 9 weeks of age were inoculated by intraperitoneally injecting EMC virus M variant (10⁴TCID₅₀/0.1 ml/animal) which is passaged in mice. The mice which tested positive for glycosuria and hyperglycemia were examined histopathologically 2 or 5 months after inoculation. The kidneys were examined for thickening of Bowman's capsule and the mesangial matrix. These changes were more clearly observable 5 months after inoculation than they were 2 months after inoculation.

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In this study, the architecture of the interstitial tissue of the boar testis was examined by using scanning and transmission electron microscopes. The boar testis was remarkable for the abundance of interstitial tissue, and Leydig cells having many microvilli in their surface were almost round in shape. Both bundles of collagen fibers and networks of reticular fibers were observed around the Leydig cells.The capillary in the interstitial tissue of the boar was a muscle type, and both pericytes and collagen fibers were observed around the capillaries. The lymphatic capillary was poorly developed in the interstitial tissues of the boar testis. Endothelial cells were the only component of the capillary wall, and anchoring filaments were often observed on the abluminal surface of the endothelium.

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We tried to make a successful collection of semen by human fingers, using ten African green monkeys (Cercopithecus aethiops) aged from 5-7 years, which were bred and reared at Tsukuba Primate Center for Medical Science. Under anesthesia, the animals were laid side way and their penes were gently stimulated by fingers. Semen was successfully obtained from five animals aged 6-7 years, but it was impossible to collect semen from the remaining five animals aged 5 years. It took only 5-15 seconds from the beginning of stimulation to the end of semen collection. The volume of semen collected, the number of spermatozoa and spermatozoal motility rate averaged 1.5ml, 3.1 x 10⁸/ml, and 46%, respectively.

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Spermatozoa from cauda epididymis of mature mice were suspended in preservation solution (Dulbecco's PBS containing raffinose in combination with glycerol, DMSO or skim milk as freezing protective agents) . The suspension was frozen by the dry ice-alcohol method and preserved for 1-120 days in liquid nitrogen (-196°C) . Highest sperm viability after thawing was obtained with a conbination of 10% raffinose and 5 % glycerol or with a conbination of 10% raffinose and 10% DMSO. These frozen thawed sperm were found to have fertilizing capacity when used forin vitrofertilization. The 2-cell embryos obtained through the above procedures developed into normal pups at a high rate when transferred into the oviducts of pseudopregnant female mice.

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A survey on the number of animals used in experiments including bioassay, diagnosis, education and preparation of biological agents such as vaccine between April 1988 and March 1989 was conducted. Out of 823 universities, institutes, testing laboratories and companies, 579 replies were received. The distribution of the number of animals is shown in the following tables:

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