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Exposure to novel objects typically evokes avoidance behavior in wild animals, which is called neophobia. We previously found that wild brown rats (Rattus norvegicus) that were trapped in a park in downtown Tokyo, Japan, exhibited neophobia. We also found that this behavior was accompanied by the activation of the basolateral complex of the amygdala (BLA). Previous studies have suggested that genetic factors are the primary determinants of neophobia. Since rats in cities form populations with distinct genetic characteristics, it is reasonable to assume that wild rats caught at different locations in urban centers will exhibit different levels of neophobia. Here we assessed the intensity of neophobia in wild rats trapped at a wholesale market in Tokyo. Although we performed exactly the same experiment in which neophobia was observed in wild rats trapped at the park, the presence of novel objects did not affect the behaviors of wild rats trapped at the market. Conversely, laboratory rats showed approach and exploratory behaviors as seen in the previous study, suggesting that the experiment was performed appropriately. Compared to the laboratory rats, the lack of behavioral changes in the wild rats was accompanied by fewer Fos immunoreactive cells in the BLA. In addition, the numbers of Fos immunoreactive cells in the bed nucleus of the stria terminalis and ventromedial hypothalamus were similar between the two types of rats. The results demonstrated the existence of wild rats that were indifferent to novel objects.

Undesirable behaviors exhibited by cats are regarded as feline behavioral problems. Like canine behavioral problems, feline behavioral problems are commonly reported by owners. Thus, in order to advise cat owners appropriately, veterinarians, particularly general veterinary practitioners, are encouraged to have sufficient knowledge of these problems. However, in ordinary Japanese homes, only a small number of feline behavioral problems have been investigated in terms of prevalence and relevant factors. Comprehensive survey data may improve understanding of feline behavioral problems. In the present study, we distributed an online survey including general information and questions about 17 feline behaviors which can be problematic for owners, to investigate: (1) the prevalence of feline behavioral problems, (2) the most common behavioral problems, and (3) relevant factors for each behavior. We collected 1,376 valid responses. Among all cats, 75.7% exhibited at least one behavioral problem. "Pica" and "Showing fear while on the examination table" were the most frequently reported behavioral problems. To identify factors related to the expression of each behavior, we conducted multiple logistic regression analyses, with the presence of behaviors as objective variables and cats' general information as explanatory variables. Age, sex, breed, number of cats in the household, and type of residence were each associated with more than one behavior. The present study revealed the prevalence of 17 feline behavioral problems in Japanese homes and relevant factors of each behavior. These findings will help veterinarians understand and address feline behavioral problems.

Wild animals tend to avoid novel objects that do not elicit clear avoidance behaviors in domesticated animals. We previously found that the basolateral complex of the amygdala (BLA) and dorsal bed nucleus of the stria terminalis (dBNST) were larger in trapped wild rats compared with laboratory rats. Based on these findings, we hypothesized that the BLA and/or dBNST would be differentially activated when wild and laboratory rats showed different avoidance behaviors towards novel objects. In this study, we placed novel objects at one end of the home cage. We measured the time spent in that half of the cage and expressed the data as a percentage of the time spent in that region with no object placement. We found that this percentage was lower in the wild rats compared with the laboratory rats. These behavioral differences were accompanied by increased Fos expression in the BLA, but not in the dBNST, of the wild rats. These results suggest that wild rats show greater BLA activation compared with laboratory rats in response to novel objects. We also found increased Fos expression in the paraventricular nucleus of the hypothalamus, ventral BNST, and ventromedial hypothalamus, but not in the central amygdala of wild rats. Taken together, our data represent new information regarding differences in behavioral and neural responses towards novel objects in wild vs. laboratory rats.

Undesirable behaviors exhibited by dogs are called canine behavioral problems. Behavioral problems are common among dogs kept in ordinary homes; hence, veterinarians need to have sufficient knowledge of these problems, so as to advise owners. Among dogs kept in ordinary Japanese homes, only a few behaviors have been investigated regarding prevalence and relevant factors. To improve understanding of behavioral problems, a comprehensive survey is crucial. In this study, we distributed an online survey including general information and questions about 25 canine behaviors that can become problematic to owners, to investigate (1) the prevalence of behavioral problem, (2) the most common behavioral problems, and (3) the relevant factors of each behavior. We collected 2,050 valid responses. Among all dogs, 86.0% exhibited at least one behavioral problem. "Barking at noises inside the house", "Barking at unfamiliar visitors", and "Pica" were the most frequent behavioral problems. To reveal those factors related to the expression of each behavior, we performed multiple logistic regression analyses, with the presence of behaviors as objective variables and dogs' general information as explanatory variables. Breed, age, the presence of other dogs in the household, sex, neutering status, source of acquisition, and type of residence were associated with 19, 16, 5, 4, 4, 4, and 1 behaviors, respectively. This study revealed the prevalence of 25 canine behavioral problems in Japan and relevant factors of each behavior. Our findings will be useful for veterinarians in advising owners about preventing behavioral problems.

Wild animals typically exhibit defensive behaviors in response to a wider range and/or a weaker intensity of stimuli compared with domestic animals. However, little is known about the neural mechanisms underlying "wariness" in wild animals. Wild rats are one of the most accessible wild animals for experimental research. Laboratory rats are a domesticated form of wild rat, belonging to the same species, and are therefore considered suitable control animals for wild rats. Based on these factors, we analyzed structural differences in the brain between wild and laboratory rats to elucidate the neural mechanisms underlying wariness. We examined wild rats trapped in Tokyo, and weight-matched laboratory rats. We then prepared brain sections and compared the basolateral complex of the amygdala (BLA), the bed nucleus of the stria terminalis (BNST), the main olfactory bulb and the accessory olfactory bulb. The results revealed that wild rats exhibited larger BLA, BNST and caudal part of the accessory olfactory bulb compared with laboratory rats. These results suggest that the BLA, BNST, and vomeronasal system potentially contribute to wariness in wild rats.

Wild animals generally avoid even small and harmless novel objects and/or familiar objects moved to a novel position, which is termed "new-object reaction". Although new-object reaction appears to be a biologically important characteristic for animals, little progress has been made in understanding the neural mechanisms underlying new-object reaction. One reason might be the lack of effective experimental animals. Two strains of roof rats (Sj and Og strains) were established from wild roof rats caught in Shinjuku, Tokyo and one of the Ogasawara Islands, respectively, by a Japanese pest control company. Based on the rat caregivers' informal observations, we conducted behavioral and anatomical tests to assess the validity of Sj and Og strains for the analyses of new-object reaction. In Experiment 1, the Sj strain showed reduced food consumption compared with the Og strain when food was provided in a novel way, suggesting that the Sj strain had a stronger avoidance of novel objects compared with the Og strain. Experiment 2 demonstrated that the basolateral complex of the amygdala and bed nucleus of the stria terminalis in experimental Sj rats had a larger percentage area compared with that of experimental Og rats, indicating these nuclei might be involved in the difference observed in avoidance of novel objects between the strains. Taken together, the present study suggests that Sj and Og strains are effective experimental animals for assessing new-object reaction.

Pavlovian fear conditioning is an experimental procedure in which a conditioned stimulus (CS) acquires an ability to elicit fear responses. This type of conditioning depends on the basolateral complex of the amygdala (BLA) and/or central amygdala (CeA). We previously found that rats showed reduced fear responses to an auditory CS when they were subjected to a pre-training chemical lesion of the entire right amygdala as compared with the left amygdala. Based on this finding, we hypothesize that the BLA and/or CeA in the right hemisphere will be more strongly activated by an auditory CS than those in the left hemisphere. To test this hypothesis, we re-exposed fear-conditioned and non-conditioned rats to an auditory CS 1 day after fear conditioning. We assessed Fos expression in the BLA and CeA in each hemisphere. We found that fear-conditioned subjects showed fear responses, such as increased freezing and decreased walking, as well as increased Fos expression in the BLA and CeA. When we compared Fos expression between hemispheres, Fos expression in the CeA, but not the BLA, was greater in the right hemisphere compared with the left hemisphere. These results suggest that the right CeA is more strongly activated by the auditory CS.

Eighteen autosomal microsatellite loci were examined using 275 Shiba Inus in Japan. Eighteen dogs representing eight trios were obtained from four breeders to calculate mutation rates, and 257 dogs kept by owners were collected through veterinary clinics throughout Japan to calculate population genetic parameters and estimate discrimination power. After two loci (INU005 and AHTk253) were excluded, average expected heterozygosity (He), polymorphic information content (PIC) and fixation index (F) were 0.665, 0.623 and 0.046, respectively. The combined power of discrimination over the 16 microsatellite markers was more than 0.9999. Therefore, it is suggested that these 16 microsatellite loci recommended by the International Society for Animal Genetics (ISAG) are applicable for individual identification and parentage testing of domestic Shiba Inu in Japan.

A 13-year-old spayed mixed-breed dog was diagnosed with a gastrointestinal stromal tumor (GIST) after histopathological examination of an abdominal mass. Five months after surgical resection of the tumor, we detected the recurrence of GIST with multiple disseminated abdominal lesions. A sequence analysis of cDNA obtained from a biopsy of the recurrent tumors revealed a mutation within exon 9 of the c-kit gene (1523A>T, Asn(508)Ile), which has been shown to cause ligand-independent phosphorylation of the KIT protein in GISTs and canine mast cell tumors (MCTs). Upon detection of the recurrent tumors, we initiated treatment with imatinib mesylate (10 mg/kg, q 24 hr). After 2 months, the dog achieved complete remission. Our findings indicate that canine GIST, and possibly MCT, may be responsive to molecular-targeted therapy.

In sheep and goats, exposure of seasonally anestrous females to males or their fleece/hair activates the gonadotropin-releasing hormone (GnRH) pulse generator leading to pulsatile luteinizing hormone (LH) secretion. Pheromones emitted by sexually mature males are thought to play a prominent role in this male effect. In the present study, we first aimed to clarify whether the male goat pheromone is effective in ewes. Seasonally anestrous St. Croix ewes were exposed to hair extracts derived from either intact or castrated (control) male Shiba goats. The male goat-hair extract significantly increased LH secretion compared to the control, suggesting that an interspecies action of the male pheromone occurs between sheep and goats. Using the male goat-hair extract as the pheromone source, we then aimed to clarify the neural pathway involved in the signal transduction of the male pheromone. Ewes were exposed to either the goat-hair extract or the control and sacrificed 2 hr after the exposure. Expression of c-Fos, a marker of neuronal activation, was immunohistochemically examined. The male goat-hair extract significantly increased the c-Fos expression compared to the control in regions of the vomeronasal system, such as the accessory olfactory bulb and medial amygdala, and the arcuate nucleus. The main olfactory bulb did not exhibit any significant increase in the c-Fos expression by the male goat-hair extract. This result suggests that the neural signal of the male pheromone is conveyed to the GnRH pulse generator through the activated regions in ewes.

Canine aggression is one of the behavioral problems for which veterinary behaviorists are most frequently consulted. Despite this, the classification of canine aggression is controversial, and there are several classification methodologies. While the etiology of canine aggression differs among the types of aggression, the behavioral background underlying aggression is not well understood. Behavior trait-based evaluation of canine aggression would improve the effectiveness and efficiency of managing canine aggression problems. We developed a questionnaire addressing 14 behavioral items and items related to four types of canine aggression (owner-, child-, stranger- and dog-directed aggression) in order to examine the associations between behavioral traits and aggression in Shiba Inu. A total of 400 Shiba Inu owners recruited through dog events (n=134) and veterinary hospitals (n=266) completed the questionnaire. Factor analysis sorted the behavioral items from both the event and clinic samples into four factors: "sociability with humans," "reactivity to stimuli," "chase proneness" and "fear of sounds." While "reactivity to stimuli" correlated significantly positively with all of the four types of aggression (P=0.007 to <0.001), "sociability with humans" correlated significantly negatively with child- and stranger-directed aggression (P<0.001). These results suggest that the behavioral traits involved in canine aggression differ among the types of aggression and that specific behavioral traits are frequently simultaneously involved in several types of aggression.

Guide dogs help visually impaired persons both physically and psychologically. More than half of all candidate dogs do not qualify, mainly for behavioral reasons. Improved training efficacy is desirable, and earlier prediction of qualification-related traits would be beneficial. In a previous study, we identified 'Distraction', assessed during the training period, as an important behavioral trait for judging the qualification of guide dogs at the Japan Guide Dog Association. As a second step, we aimed to develop an index that can predict during the puppy period. In this study, candidate guide dogs, 5-month-old Labrador retrievers, were assessed by puppy raisers using a newly developed questionnaire that consisted of 20 items. The same dogs were assessed later, at 15 months, by trainers to determine 'Distraction'. In principal components analysis, nine items, including excitability toward strangers, initiative while out for a walk, and exploration, composed the first principal component (PC1). When we compared PC1 points with 'Distraction' points, the two categories were positively correlated (n=110, r(s)=0.31, P=0.0009). Although the accuracy of the questionnaire should be increased, the results of the present study suggest that it may be possible to assess and predict 'Distraction', which is associated with disqualification for guide dogs, early in the puppy-raising period.

Previously, we have demonstrated that the alarm pheromone deteriorates sexual behavior in male rats, which was blocked by pretreatment with a corticotrophin-releasing hormone (CRH) antagonist. Studies have shown that an opioid antagonist blocked the deterioration of male sexual behavior following intracerebroventricular administration of CRH. Therefore, possibly, the pheromone effects could also be mediated by the opioid system. In this study, we pretreated rats with naloxone, an opioid receptor antagonist, and examined the following sexual behavior modulations in male rats that were exposed to the alarm pheromone. Naloxone blocked the deterioration of sexual behavior in a dose-dependent manner. On the basis of these data and the results of the previous study, we conclude that the alarm pheromone activates the CRH system, which then activates the opioid system, to deteriorate male sexual behavior.

A Japanese domestic long-hair cat of about 8 years of age was presented with vomiting and hematochezia and was found to have significant hypereosinophilia. Bone marrow aspiration revealed moderate increases of eosinophilic lineages. Histopathological examination revealed mild eosinophilic and epitheliotropic T-lymphocytic infiltrations in the duodenum. Although the cat remained asymptomatic with only prednisolone administration, the cat presented with hematemesis, weight loss, and severe anorexia 512 days after the initial presentation. Subsequently, gastrointestinal perforation developed, and the cat died on Day 536. Histopathological examination of autopsy specimens revealed mixed cellular infiltration including eosinophils and neoplastic lymphocytes in the intestinal lymph nodes, intestine, liver, spleen, and pancreas. Immunohistochemical examination supports a diagnosis of intestinal T-cell lymphoma with severe hypereosinophilic syndrome.

Pheromones are defined as substances released from an individual (donor) that influence a second individual (recipient) of the same species. However, it is unclear whether mammalian pheromones can affect the donor itself. To address this question, the effect of self-exposure to an alarm pheromone was examined. Exposure to the alarm pheromone resulted in an enhanced anxiety response, which was not different between recipients that perceived their own pheromone and those that perceived another individual's pheromone. The present results suggest that the alarm pheromone influences the emotional system of the recipient as well as induces similar anxiogenic effects on the donor rat that released the alarm pheromone. This is the first evidence demonstrating the effectiveness of mammalian pheromone self-exposure.

Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.

The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.

Emotional behavior can be characterized by interactions between genes and environments, especially in the developmental period. However, in mice, the effects of paternal care on offspring's emotional development are not well understood even in cases in which the male mice show stable paternal care to the pups. In this study, we used a reciprocal hybrid model of C57BL/6 and BALB/c mice, which show different parental behaviors and emotional responses, and compared the emotional responses in pups in their adulthood by using the elevated plus-maze test. It was found that inbred male mice showed intensive parental behavior toward their pups, especially as parental contact behavior, and that the licking/grooming behavior of males was dependent on the strain of the female they were paired with. Also, BALB/c pairs showed less parental contact behavior than C57BL/6 pairs. In the elevated plus-maze test, BALB/c males and females showed higher anxiety behavior, and reciprocal hybrid offspring showed intermediate values between C57BL/6 and BALB/c mice. Although no significant difference was found in the parental care that offspring received and anxiety-related behavior between the two reciprocal hybrid strains, BALB/c pairs showed less parental contact behavior toward the pups as compared to C57BL/6 pairs, which would be associated with the higher anxiety-related behavior exhibited in the adulthood of BALB/c strain.

An 8-year-old miniature dachshund presented with a right femoral muscle mass and anorexia. Cytology of the mass revealed a number of small-sized lymphoid cells containing a pleomorphic-shaped dense nucleus and narrow pale cytoplasm. Histopathology indicated that neoplastic lymphoid cells proliferated in skeletal muscles and replaced the muscular architecture. Immunohistochemical and genetic examinations confirmed the diagnosis of primary skeletal muscle lymphoma classified as the pleomorphic small cell type and T-cell low-grade. Although the dog suffered at least three relapses by combination chemotherapy, the dog survived 713 days after the initial presentation.

Guide dogs for the blind help blind people physically and mentally in their daily lives. Their qualifications are based on health, working performance and temperament; approximately 70% of dogs that fail to qualify are disqualified for behavioral reasons. In order to achieve early prediction of qualification, it would be essential as the first step to identify important temperament traits for guide dogs. Therefore, we administered a questionnaire consisting of 22 temperament items to experienced trainers to assess candidate dogs at the Japan Guide Dog Association after three months of training, which was at least three months prior to the final success (qualified as a guide dog) or failure (disqualified for behavioral reasons) judgment. Factor analyses of question items stably extracted three factors with high internal consistency, Distraction, Sensitivity and Docility. When we compared factor points between successful dogs and failed dogs, the successful dogs showed significantly and consistently lower Distraction points and higher Docility points. Additionally, Distraction points could predict qualification with 80.6% accuracy and detect 28.2% of the failed dogs that had higher Distraction points than any of the successful dogs. Of the nine question items not included in the three factors, two items (;Aggression' and ;Animal interest') were consistently associated with qualification. These results suggest that Distraction is stably assessable and has the strongest impact on success or failure judgment; therefore, it will be the first target to establish a behavioral test that may lead to early prediction of guide dog qualification.

To clarify the behavioral profiles of 9 feline purebreds, 2 Persian subbreeds and the Japanese domestic cat, a questionnaire survey was distributed to 67 small-animal veterinarians. We found significant differences among breeds in all behavioral traits examined except for "inappropriate elimination". In addition, sexual differences were observed in certain behaviors, including "aggression toward cats", "general activity", "novelty-seeking", and "excitability". These behaviors were more common in males than females, whereas "nervousness" and "inappropriate elimination" were rated higher in females. When all breeds were categorized into four groups on the basis of a cluster analysis using the scores of two behavioral trait factors called "aggressiveness/sensitivity" and "vivaciousness", the group including Abyssinian, Russian Blue, Somali, Siamese, and Chinchilla breeds showed high aggressiveness/sensitivity and low vivaciousness. In contrast, the group including the American Shorthair and Japanese domestic cat displayed low aggressiveness/sensitivity and high vivaciousness, and the Himalayan and Persian group showed mild aggressiveness/sensitivity and very low vivaciousness. Finally, the group containing Maine Coon, Ragdoll, and Scottish Fold breeds displayed very low aggressiveness/sensitivity and low vivaciousness. The present results demonstrate that some feline behavioral traits vary by breed and/or sex.

Two cats showing chronic vomiting, diarrhea and weight loss were found to have leukocytosis with marked eosinophilia. Both cats were diagnosed with hypereosinophilic syndrome by the findings of increased eosinophils and their precursors in the bone marrow, eosinophilic infiltration into multiple organs, and exclusion of other causes for eosinophilia. Although cytoreductive chemotherapy with hydroxycarbamide and prednisolone was performed, these two cats died 48 days and 91 days after the initial presentation.

Tryptophan hydroxylase (TPH) serves as the rate-limiting enzyme in the biosynthesis of serotonin, and two forms of TPH genes, TPH1 and TPH2, have been reported with specific nucleotide sequences and expression patterns. We determined the sequences of these two genes and identified polymorphisms within their coding regions. We identified five novel single nucleotide polymorphisms in the TPH2 gene, one of which resulted in a non-synonymous mutation and the remaining four in synonymous mutations. Comparison of the genotype frequencies of the polymorphisms among five popular breeds with different behavioral traits revealed statistically significant interbreed differences for three polymorphisms. These results suggest that these polymorphisms would be useful genetic markers when performing behavior studies in dogs.

Glutamate decarboxylase (GAD) is the primary enzyme in the brain that catalyzes the synthesis of gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter. There are two isoforms named according to their molecular weights, GAD67 and GAD65, which are encoded by GAD1 and GAD2, respectively. To investigate the association between GAD genes and temperament in domestic dogs, Canis familiaris, we sequenced the full lengths of the coding regions of these genes and identified three single nucleotide polymorphisms (SNPs) in GAD1 and one in GAD2. When comparing genotype and allele frequencies of SNPs among five breeds with different behavioral traits, statistically significant interbreed differences were observed for three SNPs in GAD1. These results suggest that GAD1 SNPs may be useful for behavioral genetic studies in dogs.

Monoamine transporters have roles to regulate the monoamine concentrations in synaptic clefts in the central nervous system and are the targets of antidepressants and psychostimulants. They include transporters for norepinephrine, dopamine and serotonin, which are encoded by the SLC6A2, SLC6A3, and SLC6A4 genes, respectivily. We sequenced the full lengths of the coding regions of these genes for dogs and identified four single nucleotide polymorphisms (SNPs) in SLC6A2 and four in SLC6A3. One SLC6A3 SNP was non-synonymous and caused an amino acid substitution from threonine to serine. The genotype frequencies of these polymorphisms differed significantly among five breeds with different behavioral traits, suggesting that novel SLC6A2 and SLC6A3 SNPs would provide additional useful information for behavioral genetic studies in dogs.

A menigioma with polygonal granular cell proliferation in an 11-year and 8-month-old male Chihuahua is described. The tumor was observed under the dura matter of the right cerebrum. Microscopically, the tumor consisted of solid growth foci of small- or large- sized polygonal cells, with pale-stained nuclei, prominent nucleoli, and fine granular to foamy eosinophilic cytoplasm. Some of the proliferating cells contained variable amounts of cytoplasmic PAS-positive granules. Immunohistochemical analysis revealed that neoplastic cells were positive for vimentin and S-100 protein. Ultrastructurally, the neoplastic cells contained vesicular structures with a few small round-shaped bodies in the cytoplasm. We diagnosed the case as canine meningioma with granular cell appearance.

The aim of the study was to determine parameters reflecting equine anxiety trait by comparing results obtained in a behavior test and an anxiety score assessed by familiar caretakers in response to a questionnaire. In the behavior test, horses were individually led into a novel room by their caretakers and loosely tethered to decrease excessive movement using the common cross-tying technique with two leads and breakable plastic cords. The horses initially remained with their caretaker for 2 min; the caretaker then left and the subject animal was left alone for 2 min. The latency to break the plastic cords, heart rate, the number of steps and pawings were recorded. When the horses were left alone, most parameters changed significantly and some showed sexual differences. A correlation analysis revealed that anxiety score assessed by caretakers showed a negative correlation with the latency to break the plastic cord and a positive correlation with heart rate only when horses were isolated. These two were suggested feasible parameters for assessing the anxiety trait of unfamiliar horses. Both the behavioral results and the anxiety scores also indicated that females were more anxious than males. Our results suggest that it would be a useful strategy for assessing other temperament traits as well to combine behavior tests with a questionnaire survey.

To clarify the behavioral profiles of 56 pure breeds of dogs in Japan, 96 small-animal veterinarians participated in a questionnaire survey using the same criteria as in preceding studies conducted in the United States and the United Kingdom. We found significant differences among breeds in all behavioral traits examined. In addition, gender differences were revealed in terms of aggression to dogs, territorial defense, excitability, general activity, dominance over owner, destructiveness, watchdog barking, and snapping at children, which were all rated higher in males than females, whereas obedience training and housebreaking ease were rated higher in females. No gender differences were evident in playfulness, excessive barking, or affection demand. Using factor analyses, "aggressiveness", "reactivity", and "trainability" were determined to be consistent with results found in the US and UK surveys. On the basis of these factor scores, seven groups of breeds were determined by cluster analysis to compare to the US survey; 22 of the 38 breeds common with the US survey were categorized into the same groups as those in that survey. The results demonstrated differences in canine behavioral predisposition among breeds and between genders. The similarity in the results between our study and previous surveys, which involved distinct geographical locales, suggests that the genetic basis of breed-specific temperamental traits is manifested irrespective of the cultural or regional identities of the owners.

Equine anxiety trait is considered an important temperament in various situations, including riding, training, and daily care. This study examined the polymorphism of the equine serotonin transporter (SLC6A4) gene as a candidate genetic element influencing equine anxiety trait. The sequence of the coding region of this gene was highly homologous with those of other mammals, and four single nucleotide polymorphisms were found by comparing the sequences of ten genetically unrelated thoroughbred horses. Radiation hybrid mapping revealed that this gene was located 26.92 cR from neurofibromin 1 on ECA 11. Using two-year-old thoroughbred horses (n=67), the association of these polymorphisms with the anxiety trait was examined, but no significant association was identified between each haplotype of the serotonin transporter gene and the anxiety score.

Excitatory amino acid transporters (EAATs) are important for terminating glutamatergic neurotransmission and protect central nervous system (CNS) neurons from glutamatergic excitotoxicity. We selected these genes as targets that may relate to canine behavioral traits. After screening four EAAT genes (glutamate transporter-1; GLT-1, excitatory amino acid transporter 4; EAAT4, excitatory amino acid carrier; EAAC1, glutamate/aspartate transporter; GLAST) for single nucleotide polymorphisms (SNPs), we identified two silent SNPs (C129T and T471C) in the GLT-1 gene. We genotyped 193 dogs of 5 breeds and found significant variation among breeds in these two SNPs in GLT-1. The C129T polymorphism was not observed in Malteses and Miniature Schnauzers. These results suggest that polymorphisms in the GLT-1 gene may be useful markers for examining how the genetic background relates to the behavioral traits of dogs.

gamma-aminobutyric acid (GABA, 30 mg/kg) was administered to aged dogs with recent history of veterinary clinic visits (mean age: 15.3 years old) once daily for 2 weeks by mixing with food. Their owners subjectively evaluated the effects of GABA on behavioral signs often associated with aging in the dogs. Improvement in some of behavioral signs was notable without any observable adverse effects. Dogs administered with GABA tended to exhibit improvement in emotional states and signs may be caused by neurovegetable dysfunction, though effects on cognitive dysfunction syndrome were not always observed. Thus, GABA administration may be one of the effective means of improving the quality of life of aged dogs.

Dopamine and noradrenaline are catecholamine neurotransmitters that are produced by biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta -hydroxylase (DBH). As a first step to elucidate the genetic background of canine behavioral traits, we selected these genes as targets and sequenced these canine genes, and found that both were highly homologous with those of human beings. Then brain cDNAs derived from ten unrelated Beagles were used to search for polymorphisms in these genes. Four single nucleotide polymorphisms (SNPs) (C97T, G168A, G180A and C264T), one of which (C97T) will cause amino acid substitution in the TH gene, and two SNPs (C789A and A1819G), both of which will cause amino acid substitutions in the DBH gene were identified. The allelic frequencies among five dog breeds (47 Golden Retrievers, 41 Labrador Retrievers, 40 Malteses, 26 Miniature Schnauzers, and 39 Shibas) were examined and found to have significant variation between them with regards to all these SNPs, except for C97T in the TH gene and A1819G in the DBH gene. The polymorphisms of C97T and A1819G were found only in the Shiba. The present results suggest that the polymorphisms of the genes encoding catecholamine biosynthetic enzymes may become important markers for examining the genetic background of behavioral characteristics in dogs.

Two kinds of FeIFN-alpha consisting of 166 amino acids (aa) and 171 aa were expressed in Escherichia coli, and the purified proteins were tested for antiviral activity on homologous and heterologous animal cells. Crude FeIFN induced in feline cells revealed antiviral activity on both homologous and heterologous animal cells. In contrast, both types of recombinant FeIFN-alpha revealed antiviral activity only on the feline cells. All of the FeIFN-alpha subtypes showed high activity to vesicular stomatitis virus, and the three species of feline viruses belonging to different families.

Monoamine oxidase B catalytically oxidizes biogenic amines such as phenylethylamine and dopamine, and its activity is presumed to be related to particular behavioral traits. In this study, we first identified a single nucleotide polymorphism (T199C) located on the putative third exon of the canine monoamine oxidase B gene, which causes an amino acid substitution from cysteine to arginine. We then examined the allelic frequencies in five dog breeds (Golden Retriever, Labrador Retriever, Maltese, Miniature Schnauzer, and Shiba) and found significant variation among them. The present results suggest that analysis of the monoamine oxidase B polymorphism could be a useful means of elucidating the genetic background of breed-specific behavioral characteristics in dogs.

Polymorphisms of human genes encoding 5-hydroxytriptamine (serotonin) receptors (5-HTRs) are thought to be associated with psychiatric disorders and behavioral traits. In the present study, we searched for corresponding polymorphisms in the dog and compared allelic frequencies for the canine 5-HTR1B, 5-HTR2A, and 5-HTR2C genes among five canine breeds. The canine genes consisted of the following: 5-HTR1B, 1170 bp; 5-HTR2A, 1413 bp; and 5-HTR2C, 1377 bp. All of these genes were highly homologous with the human genes. We found six single nucleotide polymorphisms (SNPs) in the 5-HTR1B gene (G57A, A157C, G246A, C660G, T955C, and G1146C). Genotyping of the respective SNPs revealed that there were inter-breed variations in the genotypes and allelic frequencies for four out of the six identified SNPs, suggesting that further analyses of the polymorphisms of the 5-HTR1B gene would be useful in order to gain an understanding of the genetic background underlying the diversified behavioral traits among canine species.

Various canine breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of them are under genetic control. Although dopaminergic neurotransmission system is considered to affect animal behavior, little is known about related genes in canine. Relations between specific alleles in polymorphic regions of the dopamine receptor D4 gene (DRD4) and personality or psychiatric disorders have been reported in humans, and we first found polymorphism in exon III region of the gene in 4 canine breeds. In this study we surveyed allele frequency distribution in 23 breeds including a total of 1,535 unrelated individuals. In exon III, 8 alleles including a novel allele were identified. A group of breeds in which the alleles 447b, 498 and 549 were frequent tended toward high scores in aggression-related behavioral traits than that with frequent alleles 435 and 447a. Moreover, a polymorphism based on 24 bp insertion/deletion was found in exon I region for the first time in dogs. This information may be of use for candidate gene studies of behavioral variation in dogs.

Catechol O-methyltransferase (COMT) inactivates catecholamines and catechol-containing drugs such as L-DOPA. The common genetic polymorphism Val158Met in the human COMT gene is suspected to be associated with "persistence" or risk for schizophrenia. In this study, we attempted to identify the canine COMT gene fragment and to find a similar polymorphism and to reveal its genetic distribution among five representative canine breeds. We found that the amplified gene consisted of 663 bp nucleotides and was 84% homologous with the human COMT gene. The single nucleotide polymorphisms, guanine adenine substitution, were observed at the 39th, 216th and 482nd nucleotides. From the genotyping of the 216th polymorphism among 266 dogs by the polymerase chain reaction-restriction fragment length polymorphism method with restriction enzyme EagI, and that of the 482nd polymorphism with restriction enzyme SfcI, we found inter-breed variations of genotypes as well as of allelic frequencies for both of these polymorphic regions. These results suggest that the identified polymorphisms will be useful tools in elucidating the genetic background of canine behavioral traits.

Ultrasonic vocalizations (USVs) are observed along with freezing behavior when rats are placed under fearful situation. Coping style with stress is categorized into two forms, namely passive and emotional avoidance and active operant avoidance. In this study, fear-induced USV was compared between two conditioning protocols. Two groups of male Wistar rats were contextually conditioned for 10 days by exposing to the shock box. One group was shock-unavoidable and the other shock-avoidable by operant control of emitting the USVs. They were then proceeded to the extinction phase. As the results, the shock-unavoidable group emitted greater USV in both conditioning and extinction phases. The extinction burst, commonly seen in active learned responses, was not observed in either group. The duration of freezing was also longer in shock-unavoidable conditioned rats. These results suggest that under the shock-unavoidable condition, rats receive more stress and thus emit more USV. It is therefore concluded that fear-induced USV is an emotional and passive response to the stress, rather than being controlled in an operant-way.

Early postnatal experiences have been shown to have a tremendous influence on behavior development. In this study, we focused on weaning time and investigated the effects of early weaning on adulthood aggressive behavior in mice with the resident-intruder test. Early weaning resulted in decreased sideways threat and tail rattle. In addition, the frequency of attack bites was more variable in the early weaned group, although the mean frequency did not differ from the normally weaned group. The results of the present study suggest that aggressive behavior is also affected by early weaning manipulation.

A major constituent of the characteristic "goaty odor" 4-ethyl octanoic acid (4EOA) was previously shown to have no primer pheromone activity. This was also confirmed by our own bioassay system utilizing the recording technique of neural activity of the hypothalamic gonadotropin-releasing hormone pulse generator in goats. However, when the synthetic 4EOA solution was kept at room temperature for several months, primer pheromone activity appeared in the same solution. Headspace gas chromatography/mass spectrometry analysis revealed that there were several newly formed substances in addition to 4EOA samples with primer pheromone activity. These results suggest that 4EOA derived substance(s) but not 4EOA itself is(are) primer pheromone in goats.

Stress-induced hyperthermia is observed in animals exposed to stressful conditions. In our previous study, plant-derived fragrances such as green odor and alpha-pinene were shown to suppress this stress response in rats. In the present study, we examined the concentration-dependence of the alpha-pinene effects on stress-induced hyperthermia. Male rats carrying telemetry transmitters were transferred individually to a new cage containing bedding that had been sprayed with 0.3, 0.03, and 0.003% concentrations of alpha-pinene or control solvent. Following transfer to the novel environment, the body temperature increased significantly, and this response was clearly suppressed when the cage was scattered with 0.03% alpha-pinene only. These results suggest that the effect of alpha-pinene on stress-induced hyperthermia can be observed only at a certain concentration.

The role of monoamine oxidase has been shown to be related to some behavioral changes including aggression and cognitive dysfunction. In order to demonstrate the basic expression patterns of monoamine oxidase in the canine brain, we determined the full-length nucleotide sequences of cDNA for canine monoamine oxidase type A (MAOA) and type B (MAOB) genes that were isolated from the canine brain cDNA library. Oligonucleotide primers for PCR were constructed based on the conserved sequences reported thus far for other mammalian species. The nucleotide sequences had open reading frames of 1584 and 1563 bp for MAOA and MAOB, respectively. Both of these genes showed relatively high homology with other species in both nucleotide (> 81%) and deduced amino acid (> 85%) sequences. In Northern blot analyses MAOA mRNA was expressed broadly in various parts of the canine brain, whereas MAOB mRNA was found only in specific brain regions, such as the hypothalamus, hippocampus, brain stem and olfactory bulb. These results suggest that MAOA and MAOB mRNAs have subtype-specific expression patterns in the canine brain.

The effects of pharmacological manipulation on the extinction process of fear-induced ultrasonic vocalizations (USVs), which are considered distress calls related to anxiety, were investigated. Male Wistar rats were conditioned to emit USVs by being given repeated electrical foot-shocks while in a chamber. After 10 sessions of conditioning, the animals started to emit USVs upon mere exposure to the shock chamber without being shocked. Using these animals, the extinction process of the USVs was examined. With repeated exposure to the chamber without shocks, the USVs first increased and then gradually decreased. i.e., the extinction burst was observed. Daily intraperitoneal injections of a benzodiazepine-GABA receptor agonist diazepam (DZP; 1.0 mg/kg) or a tricyclic antidepressant clomipramine (CLM; 20 mg/kg) inhibited this extinction burst. Moreover, CLM, but not DZP, shortened the period required for extinction as compared with the vehicle-treated animals. Following the extinction phase, the emission of USVs was enhanced by the cessation of both drug treatments. These results suggest that CLM would be useful for reducing anxiety-related behaviors in the extinction process, as long as withdrawal symptoms after long-term drug treatments are taken into consideration.

Castrated goats were treated with dihydrotestosterone (DHT) for four weeks. Skin samples were collected from the head and the rump regions before and after the DHT treatment. The primer pheromone activities of these samples were assessed neurophysiologically by recording electrophysiological manifestations of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator activity. Pheromone activity was detected in both the head and rump skin samples following the DHT treatment, although the development of sebaceous glands was limited to the head region. Taken together with our previous finding that testosterone treatment results in the appearance of primer pheromone activity in the skin sample of the head region but not of the rump region. these observations suggests that the regional difference of pheromone production would be ascribed to intrinsic expression levels of 5alpha-reductase, an enzyme converting testosterone to DHT.

The feasibility of fecal steroid analysis for pregnancy diagnosis and sex determination were tested in sika deer (Cervus nippon). Feces were collected from captive sika deer in June (non-breeding season and late-pregnancy period) and October (breeding season), and also from the rectum of 24 female sika deer (19 pregnant and 5 non-pregnant females) shot as part of programs for population control in February and March (mid-pregnancy period). In mid- and late-pregnancy periods, fecal progesterone concentrations were significantly higher in pregnant female than in male and non-pregnant female deer. In October, fecal testosterone concentrations were higher in adult male deer, and no difference was found between young males and females. These results suggest that fecal steroid analysis would be a useful means for estimating pregnancy status and for detecting adult male among wild deer.

Thirty-six cases of feline urine marking problem were collected through the cooperation of veterinary practitioners in the Kanto, Chubu, and Kansai areas in Japan, for an assessment of the clinical effect of treatment with a synthetic analogue of a feline cheek gland pheromone-like product. The mean frequency of urine marking was 14.2 times/week (median, 10; range, 1-77) at pre-treatment week (preW), and decreased significantly from the first week of treatment, dropping to 4.2 times/week (median, 2; range, 0-44) at the fourth week of treatment. This effect continued until the fourth week after cessation of treatment. These 36 cases were divided into 3 groups based on the effectiveness of treatment as demonstrated in the fourth week of treatment; 37% was categorized as the totally eliminated group (urine marking was not seen), 40% as the reduced group (the frequency of urine marking was equal to or less than 50% that of the preW), and 23% as the unchanged group (the frequency of urine marking was more than 50% that of the preW). Effectiveness of treatment in these groups was 38%, 24%, and 38% at the fourth week after the cessation of treatment, respectively. The decreasing rate of urine marking was compared between cats with and without intercat aggression, and it was revealed that the frequency of marking was sustained at high level in cats with intercat aggression. These results suggest that this pheromone treatment is as effective in Japan as has been reported in other countries for solving feline urine marking problems.

A sex determination method using DNA extracted from feces has been developed for sika deer (Cervus nippon). We determined a partial sequence of the amelogenin gene of sika deer, which exists on both X and Y chromosomes with a deletion region on the Y chromosome. Based on the sexually dimorphic sequences, we designed a pair of primers which could amplify DNA fragments the lengths of which are different between males and females. PCR products were detected in 34 out of 37 fecal samples collected from captured deer and the sexes estimated by the present method were perfectly matched with the actual sexes.

Distribution patterns of corticotropin-releasing factor (CRF), [arginine8]-vasopressin (AVP) and oxytocin (OXY) neurons were examined immunohistochemically in the female goat hypothalamus. The majority of the CRF immunoreactive (-IR) cells were located in the parvocellular part of the paraventricular nucleus (PVN) with smaller population found in the magnocellular part of the PVN. CRF-IR cells were also found in the suprachiasmatic nucleus, the preoptic area and around the fornix in the caudal part of the hypothalamus. AVP- and OXY-IR cells were similarly distributed in the hypothalamus. The majority of AVP- and OXY-IR cells were observed in the magnocellular part of PVN and the supraoptic nucleus. Smaller numbers of AVP- and OXY-IR cells were found in the parvocellular part of the PVN and lateral hypothalamic area. AVP-IR but not OXY-IR cells were located in the suprachiasmatic nucleus. CRF-IR fibers were concentrated in the external palisade zone of the median eminence (ME) with a few fibers found in the internal palisade zone of the ME, whereas AVP- and OXY-IR fibers were concentrated in the internal palisade zone of the ME with a few fibers found in the external zone. These results support the view that not only CRF but also AVP and OXY are released into the hypophysial portal blood and involved in the control of pituitary endocrine function in ruminant species.

Ovariectomized goats were implanted with the electrode arrays for monitoring the electrophysiological manifestation of the activity of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, namely multiple-unit activity (MUA) volleys associated with pulsatile luteinizing hormone secretion. They were then subjected to i.v. challenges of lipopolysaccharide (LPS) at the dose of 200 or 400 ng/kg. The interval between the MUA volleys was significantly prolonged by higher dose of LPS whereas neither amplitude nor duration of the MUA volleys was altered. These results suggest that immunological disturbance as evoked by LPS administration directly affects the hypothalamic GnRH pulse generator by slowing down the pulse frequency, and thereby lowers gonadotropin secretion from the anterior pituitary gland, which would culminate in gonadal suppression.

To establish an adequate experimental model for the study of immuno-neuroendocrine mechanisms underlying the behavioral changes during the acute infection, temporal relationship of various physiological responses to endotoxin administration was examined in ovariectomized goats. Immediately after intravenous injection of 200 ng/kg of lipopolysaccharide, there were an abrupt decrease of white blood cell number and a gradual increase of rectal temperature, which were followed by elevation of plasma levels of adrenocorticotropic hormone, cortisol, glucose, free fatty acids, and then later by an increase of heart rate. The results suggest that the endotoxin administration would evoke a stereotyped cascade of, febrile, neuroendocrine and metabolic as well as autonomic response to the activation of immune systems in the ruminant species.