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.

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.

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.

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.

Natural and subclinical Corynebacterium kutscheri infection in rats.

Distribution of Corynebacterium kutscheri was determined in 41 rats housed in a conventionally managed colony that were infected naturally and subclinically. At 2, 5, 10, 20 and 25 months after initial isolation of C. kutscheri, attempts were made to isolate C. kutscheri from 17 sites, with a new selective medium, FNC agar. In total, the prevalence (97.6%) of C. kutscheri isolation was significantly (P < 0.001) higher than the frequency (70.7%) of antibody detection. None of the rats manifested any distinct clinical signs of disease and macroscopic lesions caused by C. kutscheri were not detected. In 40 rats with subclinical infection, the organisms were most frequently isolated from the oral cavity, esophagus, cecal contents, and colon and rectum (> 95.0%). The isolation rate was next highest in the trachea, submaxillary lymph nodes, and nasal cavity (47.5 to 52.5%). The organisms hardly colonized the lung, liver, and kidney. Mean numbers of organisms found in the esophagus, cecal contents, and colon and rectum ranged from 10(3.9) to 10(4.2) CFU/g, and were significantly (P < 0.05, P < 0.01) high in comparison with those in the lung. These results indicated that many healthy rats in the naturally infected colony harbored C. kutscheri, and the organisms colonized the oral cavity, esophagus, cecal contents, and colon and rectum most frequently.

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)

[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.

[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.

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.

Effects of naloxone and naltrexone on receptive and proceptive behaviors in female rats.

The sexual receptive and proceptive behaviors induced by opiate antagonists, naloxone and naltrexone in estrogen-primed ovariectomized rats were observed under the presence of sexually active males. The females were treated intraperitoneally with naloxone or naltrexone at doses ranging from 0.5 to 4.0 mg/kg and the sexual behavior of females was tested before and after the injection of drug. The results obtained suggest that the opiate antagonists play a role in the regulation of lordosis behavior, but not proceptive behavior in female rats.

[Improvement of pituitary homograft under the kidney capsule in mice].

The model of hyperprolactinemia induced by pituitary homografts under the kidney capsule has been used mainly in the field of reproductive physiology. The authors report an improved method for pituitary grafting in mice. The procedure was as follows: 1. The male pituitary glands with normal saline were aspirated into a polyethylene tube. 2. Two incisions were made in the kidney capsule. 3. The polyethylene tube with pituitary glands was inserted via a large incision. 4. Blowing air into the tube, the pituitary glands were left under the kidney capsule and normal saline streamed out of a small incision. Using this method, all pituitary grafted mice became pseudopregnant.

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.

Ejaculations induced by p-chloroamphetamine (PCA) in rats, hamsters and mice.

The ejaculatory response induced by p-chloroamphetamine (PCA) in male rats, hamsters and mice was observed during 2 hours after the injection. The animals were treated intraperitoneally with PCA at doses ranging from 0.78125 to 160 mg/kg. The ED50 (effective dose in 50% of animals) values of PCA for the initiation of ejaculation in rats and hamsters were 1.3397 (1.0732-1.6725) and 0.1105 (0.0802-0.1522) mg/kg, respectively. On the other hand, no ejaculation was observed in any mice at any doses examined. So we concluded that there are species differences in the ejaculatory response, induced by PCA, among rats, hamsters and mice.

[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.

Central effects of yohimbine on copulatory behavior in aged male rats.

It is well known that yohimbine has a history of popular use because of its supposed aphrodisiac properties. The present study was done to determine whether yohimbine can modify the copulatory behavior of aged male rats. Adult male rats of the Wistar-Imamichi strain, 52 weeks of age and weighing 600-650g, were injected intracerebroventricularly with yohimbine hydrochloride (5, 10 micrograms/10 microliters/rat) or vehicle. Each male was then given the opportunity to mate with a receptive female for 30 min after administration of yohimbine or vehicle. Yohimbine produced significant decreases in the latency to initial mounting and significant increases in the number of mountings. However, there was no ejaculation in the yohimbine-and vehicle-treated males. This study is the first to clearly establish an important modulator of sexual arousal for yohimbine in aged male rats.

Copulatory behavior of sexually inexperienced male voles paired with proestrous females.

The copulatory behavior in sexually inexperienced male voles aged 9-13 weeks old was observed under dim red illumination. Proestrous females were used as stimulus voles in copulatory behavior tests. A 30-min test session was recorded. All males showed at least one ejaculation within 30 min. In addition, the pattern of copulatory behavior in male voles was compared to that reported previously in male hamsters. The frequencies of mount, intromission and ejaculation in male voles were similar to that in male hamsters but there were significant differences in the latencies of mounting, intromission, ejaculation and post-ejaculation between the two species.