Activin E, a secreted peptide encoded by the inhibin/activin βE subunit gene, is a member of the transforming growth factor-β superfamily, which is predominantly expressed in the liver. Recent reports have suggested that activin E plays a role in energy homeostasis as a hepatokine. Here, using transgenic mice overexpressing activin E under the control of the β-actin promoter, we demonstrate that activin E controls energy metabolism through brown/beige adipocytes. The glucose tolerance test and insulin tolerance test showed that the insulin sensitivity was improved in the transgenic mice. Furthermore, the mice had a high body temperature compared with wild-type mice. The transgenic brown adipose tissue and mesenteric white adipose tissue showed upregulation of uncoupling protein 1, which enables energy dissipation as heat by uncoupling oxidative phosphorylation from ATP production. Present results indicate that activin E activates energy expenditure through brown/beige adipocyte activation, suggesting that activin E has high potential for obesity therapy.

The spinal nerves supplying the wing membranes of Japanese little horseshoe bats, Rhinolophus cornutus were studied. The wing membrane was innervated by nerve branches of the radial, ulnar, and median nerves, showing that the membrane was formed from the skin of the forelimb rather than that of the thoracolumbar skin. The radial nerve was mainly composed of the ventral rami of C7–T1, the ulnar nerve by C8–T2, and the median nerve by C8–T1. These components of R. cornutus tended to be from a narrower range of spinal nerves and to position more caudally than those of humans. In addition, the ulnar nerve showed a distribution pattern different from that of other mammals.

Phosphofructokinase-1 (EC:2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key regulatory enzyme of glycolysis. Mammalian PFK-1 isozymes are composed of three kinds of subunits (PFK-M, -L, and -P), with different properties. It has been suggested that the proportion of PFK-1 subunits in different organs is based on the organ energy metabolism. In this study, we analyzed the activity and subunit composition of canine PFK-1. We found that, in dogs, the skeletal muscle only has PFK-M, the liver mainly has PFK-L, and the brain expresses all of them. The knowledge of the composition of PFK-1 could provide useful information for determination of the differences in glycolysis in various organs of dogs.

Maintaining a good ventricular systolic function is important in the long-term therapy of dogs with supraventricular tachyarrhythmia (SVTA). The objective of this study was to evaluate the inhibitory effect of telmisartan on myocardial injury and the resulting ventricular systolic dysfunction in a canine model of SVTA. A total of 14 dogs were randomly assigned to a Telmisartan (oral telmisartan, 1.0 mg/kg daily, n=7) or a Control (no drug administration, n=7) group; the duration of rapid atrial pacing (RAP) was 3 weeks for both groups. The cardiac troponin I (cTnI) concentration in the Control group was significantly increased after 3 weeks compared to that before RAP initiation (baseline), but no significant difference was observed in the Telmisartan group. Moreover, the cTnI concentration at 3 weeks was significantly lower in the Telmisartan group than in the Control group. The left ventricular fractional shortening was significantly decreased at 3 weeks compared to that at baseline in both groups. However, fractional shortening at 3 weeks was significantly higher in the Telmisartan group than in the Control group. The cardiac output values in the Control group were significantly decreased at 3 weeks compared with those at baseline, but no significant difference was observed in the Telmisartan group. This study demonstrates that telmisartan inhibits the reduction in ventricular systolic function and prevents myocardial injury in a canine model of SVTA. Therefore, telmisartan is suggested as a novel treatment for canine SVTA.

A definitive diagnosis of focal liver lesions (FLLs) requires invasive procedures for histopathologic examination. Thus, a simpler noninvasive diagnostic method, such as conventional ultrasonography combined with clinical data, is needed for the prediction of liver malignancy. The objective of this study was to examine the diagnostic accuracy of clinical data and ultrasonographic (US) features to differentiate benign and malignant liver lesions. Medical records and US images from dogs with FLLs that underwent abdominal US and histopathologic examinations following surgery or liver biopsy were retrospectively reviewed. Clinical data, including signalment, clinical signs and laboratory findings, and the US features of liver lesions that could act as predictive factors were assessed using univariate and multivariate analyses to evaluate the associations between predictive factors and liver malignancy. Based on the histopathologic results, 55 dogs with malignant lesions and 28 dogs with benign lesions were included in the study. The results of univariate analysis showed that several US features and platelet count were significantly associated with liver malignancy. Multivariate analysis revealed that the platelet count (thrombocytosis; odds ratio [OR]: 4.13, 95% confidence intervals [CI]: 1.81−9.41), lesion size (4.1 cm or greater; OR: 23.83, 95% CI: 3.74−151.95) and echotexture of FLLs (heterogenous; OR: 8.44; 95% CI: 1.37−51.91) were independent predictors for differentiating benign and malignant liver lesions, suggesting that a combination of clinical data and US findings of FLLs could predict liver malignancy in dogs.

The purpose of this study was to clarify the distribution of marbofloxacin (MBFX) within the bronchoalveolar region of calves. Four clinically healthy calves were intramuscularly injected with a single dose of MBFX (2 mg/kg). Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained for each calf at 0 (before administration), 1, 2, 6 and 24 hr after injection of MBFX. The injections and series of sample collections were conducted and repeated again after two weeks. The results show that the MBFX concentrations in the pulmonary epithelial lining fluid (ELF) were significantly higher than that in plasma and in alveolar cells at 2 hr after injection (P<0.05). For concentrations of MBFX within the ELF, the mean area under the MBFX concentration curve calculated during the 0 to 24 hr timeframe (AUC_0–24) was significantly higher than the mean determined from samples collected from the plasma (P<0.05). These results suggest that intramuscularly injected MBFX was well distributed in the bronchoalveolar region.

Beta-blockers are used to treat cats with hypertrophic obstructive cardiomyopathy (HOCM). However, there are various hemodynamic responses to beta-blockers. This retrospective study aimed to explore the relationship between the response to carvedilol and the presence of geometric abnormalities. Medical records were reviewed for 16 cats diagnosed with HOCM. Cats were divided into two groups based on the velocity of the left-ventricular outflow-tract after carvedilol treatment (responder: eight cats, non-responder: eight cats). Baseline intergroup comparison revealed that anterior mitral valve leaflet length and diastolic left-ventricular posterior-wall thickness were significantly greater in the non-responder group. Longer anterior mitral valve leaflet and thicker left-ventricular posterior-wall may cause poor response to carvedilol. Thus, these properties may predict a lack of response to carvedilol therapy.

The present study was conducted to reveal characteristic features of albino large rabbit (JW-AKT) which we formerly established a specific pathogen-free (SPF) colony. Body weights of JW-AKT rabbit at 52 weeks old was 5.7 ± 0.4 kg in males and 6.4 ± 0.4 kg in females. Weight of body, heart, lung and kidney in JW-AKT rabbit was significantly higher than in Japanese white and New Zealand white rabbits in both sexes. Though the body weight (BW) was rather lower in males, body length and brain weights tended to be higher in males than in females. Since body fat was significantly higher in females, what affects difference in BW is body fat, rather than the physical constitution of female JW-AKT rabbit. No critical sex difference was found in hematological parameters in JW-AKT rabbit. The results indicated that JW-AKT were about 1.5 times larger than the general laboratory rabbits with common properties in hematology. Thus, JW-AKT rabbit could be used as a novel SPF experimental animal model with some advantages in surgical experiments or collection of large amount of biological specimen.

A captured Japanese wild boar (Sus scrofa leucomystax) fetus was dicephalic. The fetus had two heads, but one body from the cranial neck region. Computed tomography imaging revealed that the two crania merged at the occipital bone, and the vertebral bodies between the atlas and the seventh thoracic vertebra were deformed. The fetus was found to have two tongues and laryngopharynges, but its esophagus and trachea were not duplicated. Each head contained a cerebrum and cerebellum, but the brains merged at the obex of the medulla oblongata, and the cervical spinal cord had duplicated ventral clefts. The heart was composed of three atria and four ventricles. This is the first report of a dicephalus with cardiac malformation in a wild boar.

The present paper describes Felis catus papillomavirus (FcaPV) type 5-associated cutaneous mass in a Domestic Shorthair cat. Histological examination revealed multicentric epidermal acanthosis with papillomavirus-associated cytopathic changes, which progressed to a tumor lobule with intact basement membrane. An association between FcaPV-5 and the cutaneous lesions was confirmed by detection of virus antigen and genes using immunohistochemistry, polymerase chain reaction (PCR), sequencing analysis, and in situ hybridization. Based on these findings, the lesions were diagnosed as FcaPV-5-associated viral plaques and Bowenoid in situ carcinoma (BISC). To date, this is the first reported case of FcaPV-5 infection in a cat in Japan, and the second case reported worldwide. For the first time this papillomavirus type is associated with BISC development.

This study represents cases with spontaneous neuritis of peripheral nerves in electric eels. Two electric eels were presented with abnormal swimming behavior and loss of appetite. Electric eels had extensive histopathologic lesions in the splenic and cardiac nerves. The lesions were characterized by swelling of neuronal cells, central chromatolysis and marked inflammatory cell infiltration consisting mainly of lymphocytes around the affected nerves. To the best of our knowledge, this is the first case report of spontaneous neuritis of peripheral nerves in electric eels.

A 9-year-old castrated male cat presented with sudden onset of paralysis in four limbs. Magnetic resonance imaging revealed an intramedullary lesion at C3, and lesions in the vermis and right hemisphere of the cerebellum, which were hyperintense on T2-weighted imaging. The cat was euthanized, and postmortem examination was performed. Grossly, transverse sectioning of the spinal cord revealed areas of malacia within the gray matter extending from C2 to C3. Histologically, the ventral spinal artery, the spinal intramedullary arteries and the arterioles of arachnoid in the cerebellum contained fibrocartilaginous emboli confirmed by metachromatic staining with toluidine blue. To our knowledge, this is the first report of fibrocartilaginous embolism involving both the cerebellum and cervical spinal cord in a cat.

An 8-year-old female Magellanic penguin (Spheniscus magellanicus) started to show epilepsy-like seizures. Subsequent magnetic resonance imaging (MRI) examinations did not reveal any responsible lesions. The neurological symptoms worsened at the age of 10. This penguin became recumbent and died 6 months later after the apparition of the recumbency. At necropsy, only multiple yellowish necrotic lesions in the air sacs and lungs were found. Histopathological evaluation of the brain showed a marked loss of Purkinje cells and many hypertrophied parvalbumin-positive basket/stellate cells were seen in the cerebellar cortex. Calbindin immunohistochemistry demonstrated disrupted arrangement of dendrites in the Purkinje cells. This case was diagnosed as cerebellar cortical degeneration with a very late onset and a slow progression in a Magellanic penguin.

This study aimed to investigate the pharmacokinetics of danofloxacin in red-eared slider turtle (Trachemys scripta elegans) following a single intravenous (IV) and intramuscular (IM) administrations of 6 mg/kg, using a two-way crossover study with 30-day washout period. Eight clinically healthy red-eared slider turtle weighing 410–600 g (mean 490 g) were used for the study. Danofloxacin concentrations were measured using the reversed-phase high-performance liquid chromatography. The plasma concentration-time data were evaluated by a non-compartmental method. After IV administration, the elimination half-life (t_1/2ʎz), mean residence time (MRT_0-∞), area under the concentration-time curve (AUC_0-∞), volume of distribution at steady state and total body clearance in plasma were 24.17 hr, 30.64 hr, 143.31 hr·µg/ml, 1.29 l/kg and 0.04 l/hr/kg, respectively. Following IM administration, t_1/2ʎz, MRT_0-∞, AUC_0-∞, peak concentration (C_max), time to reach C_max, and bioavailability in plasma were 32.00 hr, 41.15 hr, 198.23 hr·µg/ml, 8.75 µg/ml, 1.5 hr and 139.89%, respectively. Danofloxacin has clinically superior pharmacokinetic properties, including the complete IM absorption, slow elimination and wide volume of distribution in red-eared slider turtles. However, further pharmacokinetics/pharmacodynamics studies are necessary for the treatment of diseases caused by susceptible bacteria with known minimum inhibitory concentration values in red-eared slider turtles.

Ludwigia octovalvis extract (LOE) is a widely used traditional Chinese herbal medicine. To date, few studies have demonstrated the effect of LOE supplementation on exercise performance, physical fatigue and biochemical profile. The purpose of this study is to evaluate the potential beneficial effects of LOE extract on fatigue and ergogenic functions following physiological challenge. Male ICR mice from 3 groups (n=8 per group) were orally administered LOE for 4 weeks at 0 (vehicle), 61.5 (LOE-1X) or 307.5 (LOE-5X) mg/kg/day. LOE supplementation was able to dose-dependently increase endurance swimming time (P<0.0001) and decrease levels of serum lactate (P=0.0022), ammonia (P<0.0001), creatine kinase (P<0.0001), blood urea nitrogen (P<0.0001) and glucose utilization (P<0.0001) after acute exercise challenge. The glycogen in gastrocnemius muscle also increased with LOE treatment in a dose-dependent manner (P<0.0001). Biochemically, AST, ALT, LDH, CK, BUN, creatinine and UA levels were decreased with LOE treatment. Our study shows that 4-week supplementation with LOE increases muscle glycogen content storage to enhance exercise performance and anti-fatigue effects.

An assessment of acari (tick and mite) borne diseases was required to support development of risk management strategies in rural areas. To achieve this objective, blood samples were mainly collected from rural residents participating in hunting events. Out of 1,152 blood samples, 93 were positive against acari-borne pathogens from 12 prefectures in Japan. Urban areas had a lower rate of positive antibodies, whereas mountainous farming areas had a higher positive antibody prevalence. Residents of mountain areas were bitten by ticks or mites significantly more often than urban residents. Resident of mountain areas, including hunters, may necessary to be educated for prevention of akari-borne infectious diseases.

The present study was designed to describe the clinical presentation of abdominal hernias and to evaluate the efficacy of polypropylene mesh in repair of such affection in camels. Twenty-six dromedary camels were included in this study on the basis of clinical and ultrasonographic evidence of abdominal hernia. Factors associated with prevalence and clinical findings of hernia were presented and hernioplasty using polypropylene mesh was evaluated as a surgical intervention. Out of 26 studied camels, abdominal hernia was prevalent in Wadeh camels than other breeds (17 vs. 9, P<0.01). Camels <6 years of age exhibited more hernias than other age groups (18 vs. 8, P<0.01). Moreover, females showed a significantly higher prevalence (19 vs. 7, P<0.01) of abdominal hernia compared to males (26.9%, n=7). The sensitivity (96.8%) and specificity (93.1%) of ultrasonography (US) for diagnosing hernia were higher in comparison to clinical examination (88.3%). At 3 weeks postoperatively, the clinical index score of 26 operated camels was significantly reduced in comparison with pretreated (22 vs. 4, P<0.005). However, only 2 cases had recurrence of the hernia and 2 camels had slight swelling in situ. By the 6th month post treatment, all treated camels were completely recovered. In conclusion, the polypropylene mesh is a viable and consistent alternative effective treatment for abdominal hernias in camels. In addition, the clinical index scores and US provide a precise paradigm for diagnosis and preoperative planning for abdominal hernias in dromedary camels.

The sparing effects of tramadol and tramadol-lidocaine infusion on the minimum alveolar concentration (MAC) of sevoflurane in dogs as well as the entropy indices were investigated. Anesthesia was induced in eight young, healthy German shepherds weighing 27.6 ± 3.2 kg (mean ± SD) and maintained with sevoflurane. A standard tail-clamp technique was used to determine sevoflurane MAC during infusion with: sevoflurane alone to measure baseline MAC (MAC_B); tramadol (intravenous loading dose of 1.5 mg/kg and constant rate infusion [CRI] of 2.6 mg/kg/hr; MAC_T); and tramadol-lidocaine (tramadol CRI of 2.6 mg/kg/hr; and lidocaine intravenous loading dose of 1.0 mg/kg and CRI of 6 mg/kg/hr; MAC_TL). The state entropy (SE), response entropy (RE), and RE-SE difference were recorded 5 min prior to and during tail clamping. MAC_B was 2.4 ± 0.2%. Tramadol and tramadol-lidocaine CRI decreased MAC to 2.2 ± 0.3% and 1.7 ± 0.3%, respectively. The MAC-sparing effect of tramadol-lidocaine was greater than that of tramadol alone (8.2 ± 8.9% vs. 30.1 ± 10.7%; P<0.01). SE and RE in all subjects, and RE-SE difference in most subjects, were increased (all P<0.05) when they responded purposefully to noxious stimulation. A tramadol-lidocaine combination infusion can reduce anesthetic requirements to a higher degree than tramadol alone. Furthermore, MACentropy, MAC required to prevent increased entropy in response to a painful stimulation, and MAC of sevoflurane were similar in dogs.

The aim of this study was to compare the effects of laser acupuncture and electroacupuncture on postoperative pain and analgesic requirements in cats. In a prospective, randomized and blinded clinical study, thirty cats undergoing ovariohysterectomy were sedated with intramuscular (IM) ketamine (5 mg/kg), midazolam (0.5 mg/kg), and tramadol (2 mg/ kg). Before the induction of anesthesia, the animals were randomly distributed into three groups of ten cats each: LA: bilateral Stomach 36 (ST-36) and Spleen 6 (SP-6) acupoints were stimulated with an infrared laser; EA: bilateral ST-36 and SP-6 acupoints were stimulated with an electrical stimulus; Control: no acupuncture was applied. Postoperative analgesia was evaluated in the first 24 hr post-extubation using the Interactive Visual Analogue Scale and UNESP-Botucatu Multidimensional Composite Pain Scale. Rescue analgesia was provided with IM tramadol (2 mg/kg), and the pain scores were reassessed 30 min after the rescue intervention. If the analgesia remained insufficient, meloxicam (0.2 mg/kg IM, single dose) was administered. Data were analyzed using t-tests, the Mann-Whitney U test, and Friedman test. P<0.05 was considered significant. The pain scores did not significantly differ between the treatment groups at any time point (P>0.05). The prevalence of rescue analgesia was significantly higher in the Control group than in the LA and EA groups (P=0.033). Preoperative laser and electroacupuncture reduced the need for rescue analgesia during the first 24 hr after ovariohysterectomy.

This is the first study of using sublingual mucosae to establish a new urination passage in male cats suffering from complications from prior urethrostomy. In total, five domestic male cats afflicted with various complications, such as urethra stricture, scalding dermatitis, and urinary tract infection, after urethrostomy were included in the study. Sublingual mucosa was harvested to graft with abdominal muscles and further made into a tube connecting with the remaining urethra of the bladder at the cranial end and with the prepuce at the caudal end, serving as a new passage for urination. Postoperatively, all cats were alive and well recovered with no complications both in the oral cavity and at the surgical site. Radiography with contrast medium at two weeks and six months after surgery showed no leakage, stricture, or abnormality of the new urination passage. Moreover, all cats were able to urinate from the prepuce without any difficulties or signs of discomfort.

Six Thoroughbred racehorses with palmar process fractures of the distal phalanx were evaluated with standing magnetic resonance imaging (sMRI). In all the cases, the fractures were detectable on T1-weighted images and fat-suppressed images. Furthermore, multi-planar reconstruction images were useful for assessing the articular involvement of the fractures. Follow-up sMRI was obtainable in 3 cases, which revealed that the area of high signal intensity on fat-suppressed images decreased over time as symptoms improved. Our findings support the use of sMRI for the detailed evaluation of distal phalanx fractures including their articular involvement and the healing process.

This study investigated the direct effects of non-steroidal anti-inflammatory drugs (NSAIDs) and atrial natriuretic peptide (ANP) on canine-derived vascular endothelial cells (VECs). VECs were isolated and cultured from canine arteries and veins. The mRNA expressions of vascular endothelial growth factor receptor 2, cyclooxygenase-2, and natriuretic peptide receptor 1 were detected in the cultured VECs. The viability of the cultured VECs was reduced in a dose-dependent manner by meloxicam, carprofen, and robenacoxib. By contrast, dose escalations of ANP had only marginal influence on the viability of cultured VECs. NSAIDs may potentially serve as not only analgesic agents against cancerous and perioperative pain but also as adjuvant anti-angiogenic drugs in dogs with malignant tumors.

This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1–3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0–2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity.

We describe the successful surgical repair of third-degree perineal lacerations with rectovestibular fistulae in four cases using the single-stage modified Aanes method with purse-string suture of the vaginal vestibule and rectum. Fistulae formation and dehiscence of the repair were not observed after surgery. Two cows had five calves and one cow had four calves after surgery. The pregnant cows delivered normally. Additionally, there was no dehiscence of the suture line during the subsequent parturition. The breeding and fertility results obtained after the surgery are presented. According to the results of the present study, the modified Aanes method for the repair of third-degree perineal lacerations with rectovestibular fistulae appears to have a good prognosis for fertility following surgery in cows.

Bovine herpesvirus type 1 (BoHV-1), a member of the Alphaherpesvirinae, causes a variety of diseases, which result in significant economic losses worldwide. Envelope glycoprotein D (gD) of BoHV-1 plays an important role in viral entry into the permissive cells, and protective immune response. The fine mapping epitope on the gD will contribute to the understanding of viral pathogenesis and development of alternative vaccines against various diseases associated with BoHV-1. We previously reported the preparation of a monoclonal antibody (MAb) 2B6, which was raised by a truncated recombinant gD protein, demonstrating a neutralizing activity against BoHV-1 infection in Madin–Darby bovine kidney cells. This study described the identification of a linear B-cell epitope on gD using MAb 2B6. A series of partially overlapping gD proteins with glutathione S-transferase tag were generated to define the epitope recognized by MAb 2B6. The amino acid (aa) sequence ³²³GEPKPGPSPDADRPE³³⁷ was recognized by MAb 2B6 using Western blot with the variedly truncated recombinant proteins. Importantly, this epitope was highly conserved among the typical members of BoHV-1, indicating that the epitope may be utilized in diagnosis of diseases due to BoHV-1 infection. Furthermore, the minimal linear epitope sequence ³²³GEPKPGP³²⁹ on gD recognized by MAb 2B6 was confirmed using single-aa residue deletion mutation in carboxyl terminal. This finding not only contributes to our understanding of gD of BoHV-1 virion but also shows a potential for the development of vaccine candidates and diagnostic techniques.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL), a condition that threatens the sustainability of the livestock industry. A fluorescent loop-mediated isothermal amplification (fLAMP) assay targeting BLV env sequences was developed and used to evaluate 100 bovine blood samples. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay took 65 min, while the fLAMP assay took 8 min to 30 min from the beginning of DNA amplification to final judgement with a comparable limit of detection. The fLAMP is a potential tool for the rapid and simple diagnosis of BLV infection to supplement ELISA testing and can be used by local laboratories and slaughterhouses without special equipment.

In this study, we used multiplex polymerase chain reaction (PCR) to determine the sex of leopard cats (Prionailurus bengalensis euptilurus) in Korea. When we applied PCR using primers based on the intronic region between ZFX and ZFY, only one DNA band of 143 bp was detected in females, compared with two specific bands of 143 and 310 bp in males, indicating that the band patterns were clearly different between females and males. Since the set of primers also proved compatible with tissue and fecal samples, the results of the present study demonstrate that the present PCR could be a convenient tool for identifying the sex of leopard cats.

The Misaki horse is a Japanese native horse, known as the “feral horse of Cape Toi”. In this study, we acquired the genetic information to establish their studbook, and analyzed their genetic characteristics for conservation. We genotyped 32 microsatellites and a mitochondrial D-loop region in 77 Misaki horses (80.2% of the population). The average number of alleles, observed heterozygosity, and expected heterozygosity were 3.4, 0.509, and 0.497, respectively. A neighbor-joining phylogenetic tree of individuals was constructed. Moreover, the results suggested that Misaki horses experienced a bottleneck, but it was neither severe nor recent. In addition, three mitochondrial haplotypes were confirmed. Consequently, we clarified the genetic background of Misaki horses that have been resident at Cape Toi for a long time.