Non-targeted analyses of animal plasma: betaine and choline represent the nutritional and metabolic status.

Simple liquid chromatography-mass spectrometry (LC-MS) was applied to non-targeted metabolic analyses to discover new metabolic markers in animal plasma. Principle component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) were used to analyse LC-MS multivariate data. PCA clearly generated two separate clusters for artificially induced diabetic mice and healthy control mice. PLS-DA of time-course changes in plasma metabolites of chicks after feeding generated three clusters (pre- and immediately after feeding, 0.5-3 h after feeding and 4 h after feeding). Two separate clusters were also generated for plasma metabolites of pregnant Angus heifers with differing live-weight change profiles (gaining or losing). The accompanying PLS-DA loading plot detailed the metabolites that contribute the most to the cluster separation. In each case, the same highly hydrophilic metabolite was strongly correlated to the group separation. The metabolite was identified as betaine by LC-MS/MS. This result indicates that betaine and its metabolic precursor, choline, may be useful biomarkers to evaluate the nutritional and metabolic status of animals.

Detection of Corynebacterium kutscheri in the faeces of subclinically infected mice.

Mice were infected experimentally and subclinically with Corynebacterium kutscheri to recover the organism from mice faeces. The faeces were then cultured using selective furazolidone-nalidixic acid-colimycin agar. The number of C. kutscheri per gram of fresh faeces varied from mouse to mouse, but once established in the intestine, the organism was excreted in the faeces for at least five months. Viable bacteria were detected in most of the faecal samples, including those stored in the animal room for five days. The number of organisms in the stored faeces decreased gradually but did not differ significantly from those in the fresh faeces until they had been stored for more than three days. Many infected mice excreted between 10(4.77) and 10(5.37) colony forming units (CFU) of C. kutscheri per day in their faeces, and one mouse even excreted 10(3.74) CFU at eight weeks postinfection. These values showed little daily variation. Our present study showed that subclinically infected mice discharged the organism continuously and persistently in their faeces. Therefore, faecal samples would be useful for monitoring infection with C. kutscheri in living mice in a manner that is not stressful for the animals.

Functional natural killer T cells in experimental mouse strains, including NK1.1- strains.

Natural killer T (NKT) cells are a newly discovered subset of lymphocytes. It appears that this subset has potential as important regulators of immune responses. But because there are relatively few NKT cells in lymphoid organs and because of technical difficulties in detecting NKT cells in most mouse strains, the roles of NKT cells have not been fully identified and little attention has been paid to the roles of NKT cells in immunological experiments in which NK1.1- strains were used. To examine the existence of functional NKT cells in various strains of experimental mice, including NK1.1- strains, we utilized alpha-galactosylceramide (KRN7000) which is thought to react specifically with NKT cells. Indeed, we could confirm that early cytokine (IL-4 and IFN-gamma) secretion at 2 h after the injection of KRN7000 was dependent on NKT cells. With this in vivo system, we have successfully detected the presence of functional NKT cells in various mouse strains, including AKR/N, BALB/c, C3H/HeJ, C3H/HeN, C57BL/6, C.B-17, CBA/N, NC, NOD, SJL, W/Wv, aly/aly and aly/+. Notable increases of serum IL-4 were detected in W/Wv and aly/+ strains, and defective response of IFN-gamma in SJL mice and that of IL-4 in NOD mice were observed. This is the first report to show the functional significance of NKT cells in cytokine secretion in various mouse strains in response to a ligand for the T cell receptor of NKT cells.

Marking behavior is innate and not learned in the Mongolian gerbil.

We studied whether marking behavior in Mongolian gerbils would be innate or learned behavior. The marking behavior was defined as "animals rubbing their abdominal scent glands on small protruding objects". Between 21 and 90 days of age, Mongolian gerbils, which were kept under such conditions that they would be unable to learn this behavior, were observed at intervals of 5-15 days to find out if there were signs of the behavior or not. Six male and four female Mongolian gerbils were used for observing. Neonate Mongolian gerbils during the age of 3 to 28 days were fostered by ICR mother mice. Weaning Mongolian gerbils were then individually kept away from the others. Marking behavior was observed in 2 out of 6 males at 50 days of age and 2 of 4 females at 60 days and the mean frequency of the marking behavior for 10 min was 3.5 in the males and 5.0 in the females. These results suggest that marking behavior was innate and not learned behavior in Mongolian gerbils.

Sex differences in susceptibility of ICR mice to oral infection with Corynebacterium kutscheri.

Sex difference in susceptibility to oral infection with Corynebacterium (C.) kutscheri was experimentally studied in ICR mice. Immature (4-week-old) and adult (14-week-old) mice were inoculated with two infecting doses of C. kutscheri, and necropsied for bacteriological and serological survey 4 weeks after the bacterial infection. No macroscopic lesions at necropsy were demonstrated, except for one adult male given 10(9) bacteria. In immature mice, C. Kutscheri isolated from the oral cavity and cecum with FNC agar, were recovered in only 40.0% of female mice but in 90.0% of male mice given 10(6) bacteria (p < 0.05), and in only 55.6% of female mice but in 80.0% male mice given 10(8) bacteria. In adult mice given 10(9) bacteria, the organism were recovered in only 45.5% of female mice but in 90.9% of male mice (p < 0.05), furthermore, the mean number of organisms in the cecum of male mice harboring the organism was significantly higher than that in females (p < 0.01). Castration caused an increase in host resistance in adult male mice. These results indicated that ICR male mice were more susceptible than females, in terms of bacterial colonization in the cecum and the oral cavity, to oral infection with C. kutscheri.

Observation of marking-like behavior, marking behavior, and growth of the scent gland in young Mongolian gerbils (Meriones unguiculatus) of an inbred strain.

A marking-like behavior (defined by authors), a marking behavior, and growth of the scent glands were observed in young Mongolian gerbils of an inbred strain. In males and females, a marking-like behavior, in which animals rub their abdominal scent glands on the floor, began to be seen at the age of 19 days and could be seen in almost all the gerbils at 22 days of age during the suckling period. The frequency of this behavior was highest at 60 days of age (males: 17.9/10 min, females: 15.4/10 min) and there was no sex difference. Marking behavior, in which animals rub their abdominal scent glands on small protruding objects, began to be seen at the age of 40 days in males and 50 days in females. The frequency of this behavior tended to increase until 90 days of age in males (13.7/10 min), but the levels were low (2.5-5.0/10 min) in females. The values in the male group therefore tended to be higher than that in the female group. Macroscopic scent gland pads were clearly observed at the age of 30 days in males, but not until 45 days of age in females. At the age of 45-90 days, the length of the scent gland pad in males and females was 2.1-2.8 and 1.6-1.7 cm, respectively and the width was 0.3-0.5 in males and 0.2-0.3 cm in females. During this period, the length and depth of the pads in males were significantly greater than those in females (p < 0.05). Histological examination of the structure of the scent glands after the age of 45 days showed that the development of clusters of acinar cells in females occurred much later than that in males, but the basic structure of these glands was similar in both sexes. These results suggest that the marking-like behavior was manifested although during the period when the scent glands had not yet developed, whereas true marking behavior first occurred when the glands were moderately well developed.

Pathogenicity of Corynebacterium kutscheri in the Syrian hamster.

The pathogenicity of Corynebacterium kutscheri isolated for the first time from Syrian hamster was experimentally studied in hamsters. In hamsters given intramuscular (i.m.) or subcutaneous (s.c.) inoculation with 10 or 10(3) bacteria, neither clinical signs nor gross lesions were found. In those given 10(5) bacteria i.m., moderate proliferation of granulation tissue was found in the muscle of the inoculation region at necropsy. In the animals given 10(5) bacteria s.c., a nodular lesion was observed at the inoculation site 2 days post-inoculation (p.i.), but the nodules subsided gradually from 6 days p.i. and were unclear 10 days p.i. At necropsy, small abscesses were found in all the animals in this group. In those given 10(7) bacteria either i.m. or s.c., lesions were clearly observed at the inoculation site 1 to 10 days p.i., and a large abscess was noted at necropsy. The organisms were isolated only from the lesions in the groups. Agglutinating antibody in the sera was detected only in the animals given 10(5) or 10(7) bacteria. This suggests that 10(5) of C. kutscheri are needed to form localized nodular abscesses in Syrian hamsters.

Comparison of etiological and immunological characteristics of two attenuated Erysipelothrix rhusiopathiae strains of serotypes 1a and 2.

Two acriflavine-fast attenuated Erysipelothrix rhusiopathiae strains Koganei 65-0.15 of serotype 1a (strain Kg-1a) and 2 (strain Kg-2) were comparatively characterized. Biochemical characterization showed the similar reactions with slight variation between the strains. Strain Kg-2 was more resistant to acriflavine dye than strain Kg-1a. Pathogenicity of strain Kg-2 was higher than strain Kg-1a in mice of strains ddY. C3H/He and A/J. Significant differences of clinical signs between strains Kg-1a and Kg-2 were observed in occurrence of arthritis (P < 0.05) and systemic signs (P < 0.01) of only ddY mice. C3H/He mice was more resistant than ddY and A/J mice to the infection of strains Kg-1a and Kg-2. Three culture fractions, whole culture: WC, culture filtrate: CF and killed cells: KC, of strain Kg-2 were more protective than those of strain Kg-1a in ddY mice. CF of strain Kg-2 was most protective in all fractions. Heating at 56 degrees C and 100 degrees C or treatment with trypsin completely reduced the protective activity of WC of the two strains, indicating that major protective antigens of WC were protein. The present results demonstrated that immunogenicity and pathogenicity for mice were different between the two attenuated strains.

A simple method for intravenous injection and blood collection in the chinchilla (Chinchilla laniger).

A simple method is outlined for intravenous injection and blood collection from a peripheral vein in the chinchilla. Seven peripheral vein sites were successfully venipunctured in unanaesthetized chinchillas: the femoral, cephalic, auricular, saphenous, dorsalis penis, lateral abdominal and tail veins. The femoral and cephalic veins were found to be the best sites for injection and blood collection.

[Early detection of pregnancy of the rat].

A manual palpation method has been devised for early detection of pregnancy of the rat. One hand gently restrains the animal by holding it from behind in the usual manner. The thumb of the other hand is placed on the posterior abdomen and the first three fingers on the ventral midline. The three fingers were pressed gently upward and laterally. The embryos can then be palpated through the abdominal wall between the thumb and the three fingers. The number of fetuses was not affected by this palpation method. Using this technique, pregnancy was detected on the 5th day of gestation with an accuracy rate of 80% and between the 6th and 9th days with a 100% accuracy.

Changes in the estrous cycle and number of ovulated and fertilized ova in aging female IVCS mice.

IV CS mice (ddN origin) aged 90 days (control) and 180, 210, 240, 270, 300, 330, 360 and 420 days were used for observation of the length of the estrous cycle, and the numbers of ovulated and fertilized ova. The estrous cycle between the ages of 90 and 330 days presented a regular 4-day pattern. Thereafter, regularity of the cycle declined steadily between the ages of 360 and 420 days. All mice over 420 days old exhibited cessation of cyclicity and ultimately persistent diestrus. The average number of ova ovulated in mice aged between 90 and 240 days was consistent (11.8 to 11.4 ova), whereas a steady decline was observed for mice between 270 days old (10.5 ova) and 360 days old (4.8 ova). Ova were not recovered from oviducts of mice aged about 420 days. The average number of fertilized ova (2-cell stage) in mice between 90 and 210 days old showed no significant change (11.5 to 10.3), whereas the number began to decrease in mice aged between 240 days (8.7 fertilized ova) and 330 days (2.5 fertilized ova). No fertilized ova were recovered from the oviducts of mice around 360 days old. These findings demonstrate that the decline of reproductive activity is initially observed in the number of fertilized ova, followed by a decline in the number of ovulations and finally by loss of the estrous cycle.

[Selective medium for Corynebacterium kutscheri and localization of the organism in mice and rats].

A new selective medium for isolation of Corynebacterium kutscheri (CK) from animals suffering subclinical infection was made by adding furazolidone, nalidixic acid and corimycin to the heart infusion agar base, this being named FNC agar. The FNC agar inhibited the growth of gram-negative rods, Pseudomonas aeruginosa, Proteus sp. and gram-positive cocci but did not affect the growth of CK. When this medium was used to isolate CK from the oral cavity and cecal contents of mice and rats, two of 6 conventional mouse colonies and three of 8 conventional rat colonies were found to be infected, with isolation of the organism from 19 mice and 12 rats in total. The animals showed neither clinical signs nor lesions, but the antibody was positive in 11 mice and 10 rats. In mice and rats inoculated orally with 4 x 10(8) and 1 x 10(9) organisms, respectively, CK was isolated for 20 weeks post-inoculation by use FNC agar. The isolation rate of the organism was the highest in the oral cavities of both inoculated mice and rats, and also in the submaxillary lymph nodes of the inoculated rats. The organism was also recovered from the cecal contents of more than half of the inoculated mice and rats. Thus, it was considered that FNC agar was useful in isolating CK from the oral cavity and cecal contents of mice and rats with subclinical infection of the organism.

[Increase in litter size and decrease of post-implantation loss of fetuses observed in an SPF colony of Wistar-Imamichi rats].

The number of delivered offspring, corpora lutea and implantation sites was observed in an SPF colony of Wistar-Imamichi rats bred in a barrier system free of specified microorganisms and parasites and was compared with that of rats bred under conventional conditions. The litter size of 6142 SPF rats ranged from 1 to 20, averaging 12.4 +/- 0.04, a value significantly higher (P less than 0.001) than that of conventionally bred rats (11.0 +/- 0.04). Although the number of corpora lutea was also higher in SPF rats than under conventional conditions, the number of post-implantation losses in SPF rats with less than 10 offspring showed a marked decrease compared with conventionally bred rats. These results show that the number of delivered offspring is higher in SPF rats than in conventionally bred rats and indicate that the increase in litter size is due to the increase in the number of corpora lutea and decrease in post-implantation loss of embryos of fetuses.

[Comparison of the two different approaches of orchiectomy in guinea pigs].

The results of orchiectomy conducted by the abdominal and scrotum routes were compared in guinea pigs. The operation time, surgical complication and incision healing were evaluated. About operation time, there was no distinct difference between two routes. In the abdominal route, the prognosis of this case was favorable. Also the healing of an incised wound was uneventful one week after the operation. While in the scrotum route, the tunica albuginea testis and the visceral vaginal tunic are thin, so that the risks of injuring the testis and/or scattering the contents of testis seem to be high. Inflammation of the scrotum was observed in 4 out of 5 guinea pigs, that were operated by scrotum route. From these results, the abdominal route method is highly recommended in the orchiectomy of guinea pigs.

[Investigation of the development of dermatitis in hypotrichotic rats--microbiological study].

The hereditary hypotrichotic (WBN/IIa-Ht) rat is affected by dermatitis characterized by erosions and crust formation on dorsal skin areas. The bacterial flora of this dermatitis and the normal skin of the hypotrichotic rat was examined. As controls the cutaneous flora of atrichotic and Wistar rats was examined, and the results compared with those in hypotrichotic rats. The total number of bacterial colonies from the lesions and normal skin of hypotrichotic rats were 3.9 x 10(5) to 1.16 x 10(8) CFU/cm2 and 1.6 x 10(3) to 1.8 x 10(4) CFU/cm2, respectively. In the lesions, overwhelming numbers of S. aureus were detected as almost pure cultures. In the normal skin, S. aureus was predominant, accounting for about 90% of all bacteria. The total number of bacterial colonies in the atrichotic rats was the same as in the hypotrichotic rats. The majority of the isolates were Staphylococcus sp., and about half of them were identified as S. aureus. The total number of bacterial colonies from the skin of individual Wistar rats varied extremely, ranging from 64 to 2.98 x 10(5) CFU/cm2. The flora mainly consisted of coagulase-negative Staphylococcus (CNS), S. aureus, and alpha-hemolytic Streptococcus sp., and CNS was isolated most frequently. Histopathological examination of the eroded portions in hypotrichotic rats revealed many clusters of coccoid bacteria and neutrophilic cell infiltration of the epidermis. These findings suggest that the dermatitis in hypotrichotic rats was caused by S. aureus and affected by unknown traits of these rats.

Natural habitats of Corynebacterium kutscheri in subclinically infected ICGN and DBA/2 strains of mice.

Subclinically infected mice of ICGN and DBA/2 strains housed in a conventionally managed colony were examined to determine natural habitats of Corynebacterium kutscheri. At 5, 7, 9, 12 and 13 months after initial isolation of the organism from oral cavity and cecal contents of five ICGN mice, attempts were made to isolate C. kutscheri from 19 sites using a new selective medium, furazolidone-nalidixic acid-colimycin agar. From the initial survey to 13 months, C. kutscheri was isolated from 27 of 29 ICGN mice (93.1%) and 9 of 10 DBA/2 mice (90%). In contrast, antibody against C. kutscheri was detected in only 3 of 29 ICGN mice (10.3%). None of the mice manifested distinct clinical signs of infection, and only 1 ICGN mouse had macroscopic lesions such as hepatic abscess and large spleen. In 21 ICGN and 9 DBA/2 mice that harbored the organism without macroscopic lesions, the organisms were most frequently isolated from the oral cavity (ICGN:100%, DBA/2:66.7%), cecum (ICGN:95.2%, DBA/2:100%), and colon and rectum (ICGN:95.2%, DBA/2:100%). Remarkable differences between the two mouse strains were observed in colonization of the nasal cavity (ICGN:85.7%, DBA/2:0%) and trachea (ICGN:71.4%, DBA/2:33.3%). In mice of both strains, the organisms rarely colonized the lung, liver, and kidney. Mean numbers of organisms in the cecum, and colon and rectum ranged from 10(4.1) to 10(4.6) colony-forming units/g and were significantly higher in comparison with those in the small intestine (P < 0.01, P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

[Differences in susceptibility of mice among various strains to oral infection with Corynebacterium kutscheri].

Differences in susceptibility of female mice among 10 strains to Corynebacterium kutscheri infection were studied pathologically and bacteriologically. Twenty mice of each strain were inoculated orally with 4 x 10(5.0) CFU of the bacteria. The gross lesions were observed in 60.0% of BALB/c-nu/nu mice, 25.0% of CBA/N mice, 10.0% of MPS mice, and 5.0% of A/J and C3H/He mice, while BALB/cCr, C57BL/6Cr, B10.BR/SgSn, ddY and ICR mice showed neither clinical signs nor gross lesions. Six BALB/c-nu/nu, two CBA/N and one MPS mice died within 15 days after inoculation. C. kutscheri was recovered from 95.0% of BALB/c-nu/nu mice, followed by 75.0% of A/J mice, 65.0% of CBA/N mice, 55.0% of MPS and BALB/cCr mice, and 30% of C3H/He mice. On the other hand, from C57BL/6Cr, B10.BR/SgSn and ddY mice, the bacteria were recovered at less than 15.0% of the mice. No bacteria were recovered from ICR mice. C. kutscheri colonized most frequently in the cecum, colon and rectum. Number of mice having agglutinating antibodies were less than 20% and their antibody titers ranged from 1:10 to 1:80. These results indicated that there were differences in susceptibility of mice among the strains to oral infection of C. kutscheri. Namely, BALB/c-nu/nu, A/J, CBA/N, MPS and BALB/cCr mice appeared to be susceptible, and C3H/He mice intermediate, while C57BL/6Cr, B10.BR/SgSn, ddY and ICR resistant. BALB/c-nu/nu mice were most susceptible and exhibited markedly severe disease by the infection.

In vitro development of two-cell embryos obtained from female mice of different ages.

Female mice of the IVCS strain (ddN origin), aged 90, 180, 240, 270 and 300 days, were used in this experiment. The percentages of fertilized ova per ova collected from the oviducts, on Day 1 of pregnancy, of mice aged 90 and 180 days were 100% and 91.1%, respectively. Significantly decreased rates of fertilized ova were observed in mice aged 240 to 300 days as compared to 90-day-old controls (P < 0.05). Almost all two-cell embryos collected from 90- to 270-day-old mice developed into blastocysts or expanded blastocysts, and the developmental rate from 2-cell embryo to hatched blastocyst then decreased significantly (P < 0.05). The developmental rate from 2-cell embryo to hatched blastocyst also decreased significantly in 300-day-old mice (P < 0.05). These results demonstrate differences in the viability of mouse embryos collected from mice of different ages.

[Coprophagy in the germfree mouse].

Coprophagy was observed in germfree (GF) ICR mice of both sexes, and the results were compared with those of conventional mice. Frequency of coprophagy per animal per day in GF mice was 5.1 in males and 5.8 in females. In conventional (CV) mice, the frequencies were 6.2 in males and 5.3 in females (data from Zoological Science 2:249-255, 1985), with no significant differences compared with GF mice. Coprophagy in CV mice was frequently observed during 6-8 hr after lighting, whereas such close time relationships tended to weaken in GF animals. In a comparison of levels of constituents per unit weight between feces and diet, fecal crude protein and crude fat exhibited lower values than those in the diet. Levels of fecal crude ash and crude fiber were higher than those in the diet, and nitrogen-free extract was almost equal to that in the diet. No essential difference in these tendencies was found compared with CV mice. Levels of fecal vitamin B1, B2, B12 and folic acid were lower than those in the diet. In CV mice, except for vitamin B1, these vitamins exhibited either almost equal or much higher levels compared with those in the diet (data from Experimental Animals 35: 381-386, 1986). From the fact that coprophagy was observed in GF mice, it is suggested that the behavior is inherent in the mouse.

Effects of methamphetamine on copulatory behavior in male rats.

Many papers have reported the effect of amphetamine and its close relative, methamphetamine on sexual activity in humans. The effect of these drugs on animal sexual behavior is unknown. The present study was done to determine whether methamphetamine modifies the copulatory behavior of male rats. Sexually experienced rats of the Wistar-Imamichi strain, 10 weeks of age, were singly injected intraperitoneally with methamphetamine hydrochloride at doses of 0, 1, 2 or 4 mg/kg body weight. Tests were initiated at 19: 30 in the dark period. Observations were conducted for 90 min. At dosages of 1 or 2 mg/kg there were no changes in copulatory behavior ascribed to the administration of methamphetamine. At 4 mg/kg the frequencies of mounting, intromission and ejaculation were greatly decreased but the spontaneous motor activity and stereotypic behavior increased in all males. In a further experiment, males were given methamphetamine by intraperitoneal injection once a week for 8 weeks. The copulatory behavior was tested 5 times at two weeks intervals. In the 4th testing, the percentage showing ejaculation decreased. In the 5th testing, there was no ejaculation and the incidence of intromission decreased. No stereotypic behavior was displayed in any of the males during the testing. From these results, it was concluded that methamphetamine inhibits the intromitting and ejaculating behavior in male rats.