Epidemiological survey, general blood biochemistry, and histological examination of slaughtered heavy horse breeds with hemorrhage in the adipose tissue in the crest of the neck.

Fifty-four slaughtered horses were classified into groups having adipose tissue in the crest of the neck with or without hemorrhage (AH and NH groups, respectively). Blood biochemical tests (Alb, TP, T-bil, GOT, GPT, LDH, T-cho, and BUN) and an epidemiological survey (age, gender, weight, origin, breed, BCS, CNS, and hoof disease) were performed. T-bil tended to be high, while the other parameters were normal. Weight, BCS, and CNS were higher in the AH group (P<0.05). GOT was lower in the AH group (P<0.05). It was suspected that the horses in the AH group had lipomatosis. It was assumed that the adipose tissue of the horses in the AH group contained damaged capillaries, and inflammation was confirmed based on evidence of macrophages and lymphocytes.

Effect of a novel rice fermented extract on gastric ulcers in horses.

Gastric ulcers cause appetite loss, poor body condition, and colic in horses. This study investigated the protective effect of a rice fermented extract on the gastric mucosa in 17 healthy Thoroughbreds. For one month, horses in the rice fermented extract (nine horses) and control (eight horses) groups were orally administered a rice fermented extract (100%; 0.2 ml/kg, SID) and tap water (0.2 ml/kg), respectively. Gastric endoscopic images were obtained before and one month after rice fermented extract administration. The gastric ulcer score was lower after administration (median, 1; maximum, 2; minimum, 1) than before administration (median, 4; maximum, 4; minimum, 3) in the rice fermented extract group (P<0.05). In conclusion, the administration of a rice fermented extract for one month improves gastric mucosal lesions in Thoroughbreds with gastric ulcers.

Transcatheter arterial embolization in normal canine liver.

OBJECTIVE: To determine the effects of selective transcatheter arterial embolization (TAE) in the normal canine liver. STUDY DESIGN: Experimental study. ANIMALS: Adult Beagle dogs (n = 5). METHODS: Gelatin sponge particles (GSPs) were injected through a microcatheter for selective embolization of the left hepatic artery in normal dogs. Computed tomography (CT) and histology were performed during an 8-week observation period; biochemical analysis data were obtained during a 12-week observation period after TAE. RESULTS: Embolization was successful in all dogs and did not induce any change in the clinical appearance of dogs. Postoperative CT was consistent with recanalization of the artery within 2 weeks of embolization in all dogs. Hepatic enzyme levels increased temporarily after embolization but gradually returned to normal ranges. Histological examinations did not differ between treated and untreated liver tissues. CONCLUSION: TAE appears safe in normal dogs observed for 12 weeks. Arterial recanalization seems to occur within 2 weeks after injection of GSPs in the left hepatic artery. IMPACT/CLINICAL RELEVANCE: Selective TAE of the hepatic artery was well tolerated in normal dogs. Selective TAE may be applicable to canine hepatocellular carcinoma.

Novel Approach for Evaluating Pregnancy-Associated Glycoprotein and Inflammation Markers during the Postpartum Period in Holstein Friesian Cows.

We evaluated the relationship between decreased pregnancy-associated glycoprotein (PAG) levels, inflammatory parameters (serum amyloid A [SAA] and milk amyloid A [MAA]), postpartum inflammatory conditions (mastitis, ketosis, and follicular cysts), and the FOXP3 gene. Nineteen Holstein-Friesian cows were included in this study. Up to approximately eight weeks after delivery, weekly health examinations were performed for mastitis and ketosis, and reproductive organ ultrasonography was performed. The decreasing PAG rate was negatively correlated with SAA concentration (r = -0.493, p = 0.032). Cows with mastitis exhibited a slower trend of PAG decrease (p = 0.095), and a greater percentage of these cows had MAA concentrations above 12 µg/mL (p = 0.074) compared with those without mastitis. A negative correlation, although nonsignificant (r = -0.263, p = 0.385), was observed between the day-open period and decreased PAG rate. The day-open period was correlated with the presence or absence of follicular cysts (p = 0.046). Four cows that developed follicular cysts were homozygous for the G allele of the FOXP3 gene related to repeat breeders. These results indicate a relationship between a decreased PAG rate and inflammatory status during the postpartum period. Thus, suppressing inflammation during the perinatal period may improve reproductive efficiency in the dairy industry.

Relationship Between Ovary Size and Anti-Müllerian Hormone Levels in Holstein-Friesian Cows.

The aim of this study was to verify the association between ovarian size and blood AMH levels in HF cows. Sixty multiparous HF cows from three herds were included in this study. The data required for calculating the ovarian volume included the "major axis (length)," "minor axis (width)," and "thickness" of the ovary. All ultrasonography (US) images were acquired at the outermost ends/poles of both the ovaries and of the follicles (>8 mm) and corpus luteum (CL); concomitantly, the blood was sampled from the jugular or coccygeal vein. Based on the ovarian images of each cow, the following ovarian size patterns were calculated using an image analysis software: (1) total area of both the left and right ovaries, (2) individual size of the large ovary, and (3) individual size of the small ovary. For each ovary area pattern, two properties were assessed: (A) presence of follicles (>8 mm) and CL, which may not secret AMH, in the ovaries and (B) absence of follicles (>8 mm) and CL in the ovaries. Serum AMH levels were measured using enzyme-linked immunosorbent assay. The correlation between ovary size and serum AMH levels was measured in terms of the aforementioned patterns and was evaluated statistically. The results of our preliminary study with ovaries from slaughter-house cows (n = 22) revealed that the "thickness" of the ovary was not necessary for estimating ovarian volume and that length and width were sufficient. A strong correlation was observed among ovarian length, width, and thickness (r > 0.96). No significant difference was observed (p > 0.05) in the mean ages or parities among the three herds. Among the ovary sizes measured in this study, the highest correlation was found between the total size of an individual large ovary (including follicular and luteal size) and AMH levels (r = 0.387, p = 0.002). This is the first study to demonstrate the correlation between total size of individual large ovaries and serum AMH levels in HF cows. US observations of the ovaries will allow for estimation of differences in AMH levels and help predict ovarian activity and superovulation performance of cows.

Assisted hatching of poor-quality bovine embryos increases pregnancy success rate after embryo transfer.

Embryos of good, fair and poor quality were collected from superovulated cows and subjected to zona cutting (ZC) treatment using a needle under either an inverted microscope or a stereomicroscope. One (single transfer) or 2 (twin transfer) embryos with or without prior ZC treatment were transferred nonsurgically to recipients. Without the ZC treatment, lower embryonic quality resulted in lower pregnancy success rates. However, the ZC treatment increased the pregnancy success rate following transfer of poor-quality embryos, but not the pregnancy rate after transfer of good- or fair-quality embryos. No differences were observed between the pregnancy success rates after the transfer of embryos treated under the inverted microscope and those after transfer of embryos treated under the stereomicroscope, and this was the same after single and twin transfer. Moreover, ZC treatment of embryos prior to transfer did not result in an increased abortion rate, irrespective of the number of transferred embryos. In conclusion, ZC treatment improves pregnancy success rates following transfer of poor-quality embryos. Moreover, the results indicate that ZC treatment by using a stereomicroscope is practical for on-farm application.

Cystine transporter expression is a marker to identify a subpopulation of canine adipose-derived stem cells.

Adipose-derived stem cells (ADSCs) are promising cell sources for regenerative medicine due to the simplicity of their harvest and culture; however, their biological properties are not completely understood. Moreover, recent murine and human studies identified several functional subpopulations of ADSCs varying in differentiation potential; however, there is a lack of research on canine ADSCs. Cystine transporter (xCT) is a stem cell marker in gastric and colon cancers that interacts with CD44 to enhance cystine uptake from the cell surface and subsequently accelerates intercellular glutathione levels. In this study, we identified a ~5% functional subpopulation of canine ADSCs with xCT+ expression (xCTHi). Compared with those of the xCT- subpopulation (xCTLo), the xCTHi subpopulation showed a significantly higher proliferation rate, higher expression of conventional stem cell markers (SOX2, KLF4, and c-Myc), and higher expression of adipogenic markers (FABP4 and PPARγ). By contrast, the xCTLo subpopulation showed significantly higher expression of osteogenic markers (BMP1 and SPP) than xCTHi cells. These results suggest xCT as a candidate marker for detecting a functional subpopulation of canine ADSCs. Mechanistically, xCT could increase the adipogenic potential while decreasing the osteogenic differentiation potential, which could serve as a valuable target marker in regenerative veterinary medicine.

Effect of transcatheter arterial embolisation in normal canine liver using trisacryl gelatine microspheres (Embosphere).

The effect of selective transcatheter arterial embolisation (TAE) using trisacryl gelatine microspheres (TGMs) in the normal canine liver was investigated. Selective embolisation was achieved by injecting TGMs into the left hepatic artery through a microcatheter in four healthy dogs. After embolisation, computed tomography (CT), biochemical analysis and histological examination were performed during a 12-week observation period. Embolisation was successful in all four dogs. Postoperative CT revealed consistent embolisation of the artery within the experimental period in three dogs. Hepatic enzyme levels slightly increased after embolisation but tapered to normal ranges. Histological examinations revealed no abnormal changes. Thus, selective TAE with TGMs was well tolerated in normal dogs and may be applicable to canine hepatocellular carcinoma.

Therapeutic efficacy of mammary irrigation regimen in dairy cattle diagnosed with acute coliform mastitis.

The objective of this field study was to determine the therapeutic efficacy of mammary irrigation for the treatment of dairy cattle diagnosed with acute coliform mastitis caused by gram-negative bacteria. Additionally, the effects of different mammary irrigation regimen fluids such as ozone water and normal saline were compared. Dairy cattle clinically diagnosed with acute coliform mastitis (n = 57) were enrolled in the study, randomly assigned to 1 of 3 groups, and received the following treatments: systemic antibiotic administration (SAA group; n = 40), mammary irrigation regimen (MIR group; n = 10), and both treatments (MIX group; n=7). Significant antipyretic effects, as assessed by rectal temperature measurement, were observed in the MIX and MIR groups. Although 2 irrigating fluids were used, namely, ozone water and normal saline, no significant difference was observed between the 2 groups. Fourteen days after the onset of the treatments, the milk yield recovery rate in MIR group tended to be higher (p = 0.06) than that in the SAA group. Additionally, after 30 days of treatment, the MIR group cows demonstrated significantly higher successful recovery rates (p<0.05) than the SAA group cows. These results indicate that mammary irrigation with normal saline is an effective treatment for acute coliform mastitis in dairy cattle.

Canine Liver Fibrosis Model to Assess the Functions of Infused Autologous Bone Marrow-Derived Cells.

Liver cirrhosis is the end stage of chronic liver disease, and the only radical treatment for decompensated liver cirrhosis is still liver transplantation. Development of effective regenerative therapy for liver cirrhosis is an urgent task. Before human clinical trials can be considered, the safety and efficacy of any planned protocol must be confirmed in medium-to-large animals. Therefore, we have developed a novel canine liver fibrosis model for proof of concept (POC) studies.

Liver regeneration therapy through the hepatic artery-infusion of cultured bone marrow cells in a canine liver fibrosis model.

BACKGROUND: We previously reported regenerative therapies for decompensated cirrhosis based on peripheral venous drip infusion using non-cultured whole bone marrow (BM) cells, or the less invasive cultured BM-derived mesenchymal stem cells (BMSCs). Here, we assessed the efficacy and safety of hepatic arterial infusion using cultured autologous BMSCs, comparing it with peripheral infusion, using our established canine liver fibrosis model. METHODS: Canine BM cells were harvested and cultured, and the resultant BMSCs were returned to carbon tetrachloride (CCl4)-induced liver cirrhosis model canines via either a peripheral vein (Vein group) or hepatic artery (Artery group). A variety of assays were performed before and 4, 8, and 12 weeks after BMSC infusion, and liver fibrosis and indocyanine green (ICG) half-life (t1/2) were compared to those in a control group that received CCl4 but not BMSCs. The safety of this approach was evaluated by contrast-enhanced computed tomography (CT) and serial blood examinations after infusion. RESULTS: Four weeks after infusing BMSCs, a significant improvement was observed in the Vein group (n = 8) compared to outcome in the Control group (n = 10), along with a decrease in ICG t1/2. In the Artery group (n = 4), ICG t1/2 was significantly shorter than that in the Vein group at 8 weeks (Δt1/2: -3.8 ± 1.7 min vs. +0.4 ± 2.4 min; p < 0.01) and 12 weeks (Δt1/2: -4.2 ± 1.7 min vs. +0.4 ± 2.7 min; p < 0.01) after BMSC administration. Post-infusion contrast-enhanced CT showed no liver infarction, and blood tests showed no elevations in either serum lactate dehydrogenase concentrations or hypercoagulability. CONCLUSIONS: We confirmed the efficacy and safety of the hepatic arterial infusion of cultured autologous BMSCs using a canine model, thereby providing non-clinical proof-of-concept.

A new hepatic encephalopathy model to monitor the change of neural amino acids and astrocytes with behaviour disorder.

BACKGROUND/AIMS: To elucidate the pathogenesis of hepatic encephalopathy (HE), we developed a new HE model with behaviour disorder. METHODS: Male Wistar rats were divided into four treatment groups: a HE model: acetaminophen (APAP)+3-methylcholanthrene (3-MC) group (APAP+MC group); control group: acetaminophen group; 3-methylcholanthrene group; and a no-treatment group. We monitored the changes of neural amino acids in the synaptic cleft and astrocytes in the brain during behaviour disorder. RESULTS: In the APAP+MC group, alanine amino transferase, blood ammonia and glucose increased from 3 h and total bilirubin increased at 6 h. Prothrombin time was prolonged from 3 h in the APAP+MC group. The APAP+MC group exhibited centrilobular necrosis in the liver after 8 h. In the APAP+MC group, rats jumped vertically and this vertical activity increased significantly from 4 to 7 h. During the behaviour disorder, we found that glutamate and aspartate increased in the synaptic cleft from 4 h after treatment with APAP+3-MC, glutamate increased 23.9-fold at 7 h and aspartate increased 16.1-fold at 4 h, whereas glutamine did not change. At that time, we observed morphological changes of the astrocytes by immunostaining for the glial fibrillary acidic protein. CONCLUSIONS: Our new HE model demonstrated that increased excitatory neural amino acids and morphological change in astrocytes were involved in the behaviour disorder that occurs with HE.

Comparison of the amounts of endotoxin released from Escherichia coli after exposure to antibiotics and ozone: an in vitro evaluation.

Antibiotic therapy administered for treatment of gram-negative bacterial infections can exert a negative effect in the form of endotoxin shock. Therefore, a sterilization method that releases little endotoxin is required. This in vitro study aimed to compare the amounts of endotoxin released from Escherichia coli exposed to antibiotics (aminobenzylpenicillin, kanamycin, oxytetracycline, sulfadimethoxine and enrofloxacin) or ozone. The bacterial growth and amount of endotoxin released were measured at specific time points. Compared with antibiotic treatment, ozone sterilization induced release of smaller amounts of endotoxin from the bacteria. Moreover, no major differences were observed when ozone was used against the purified endotoxin. We conclude that compared with antibiotic treatment, ozone sterilization may release smaller amounts of endotoxin.

Malignant mixed tumor in the salivary gland of a cat.

The presence of a malignant mixed tumor, also known as a carcinosarcoma, in the salivary gland is very rare. Such tumors, which are typically aggressive, are characterized by the presence of carcinomatous and sarcomatous components. A 9-year-old neutered female domestic short-haired cat presented with swelling in the right mandibular lesion that had rapidly enlarged over the previous 3 weeks. Physical examination revealed a large, fluctuated and painless subcutaneous swelling that was associated with a firm mass. Radiographs of the head revealed a soft-tissue density that involved faint circular calcific opacity. Contrast-enhanced computed tomography revealed that the peripheral capsulated cystic area had a contrast enhanced region without bone lysis. The cat received a total excision of the mass and postoperative radiotherapy. Histopathological analysis of the mass revealed that it was a malignant mixed tumor. Metastasis to the lung was discovered 7 weeks later, at which time treatment was stopped.

Agenesis of hepatic lobes in a dog.

Agenesis of a hepatic lobe is an extremely rare congenital anomaly and only one dog have been reported in veterinary literature. We encountered a dog with this anomaly diagnosed by Computed tomography (CT) and portography. A two-year-old, 6.9-kg female Shih tzu dog was presented with vomiting and anorexia. The dog had no history of abdominal surgery or trauma. Biochemical analysis showed elevated plasmatic liver enzymes. CT revealed the absence of the liver parenchyma and vascular system of the left lobe, quadrate lobe and papillary process of the caudate lobe. A portosystemic shunt was also observed. The liver parenchyma and vascular system of these lobes were not detected under digital subtraction angiography during laparotomy. Furthermore, the liver parenchyma and vascular system of these lobes were not detected even when the remaining liver volume increased two months after treating the shunt vessel. CT proved itself a good option for antemortally diagnosis of hepatic agenesis in a dog.

Activity of matrix metalloproteinase-2 (MMP-2) in canine oronasal tumors.

Activity of matrix metalloprotease-2 (MMP-2) and the expression of its related molecules were examined in spontaneous canine oronasal tumors. Tissue samples from melanoma and squamous cell carcinoma possessed higher MMP-2 activity, as shown in gelatin zymography, in comparison with acanthomatous epulis and nasal adenocarcinoma. Regional lymph node invasion and distant metastases were more frequently observed in the MMP-2 positive cases. There were no significant differences by RT-PCR examination in the expression of the genes encoding MMP-2, MT1-MMP and TIMP-2 among the tumor histological types. However, the MMP-2/TIMP-2 ratio showed a significantly higher level of the genes in the malignant oral melanoma and squamous cell carcinoma. The MMP-2/TIMP-2 ratio was also positively correlated with MMP-2 activity in gelatin zymography. These results indicate that the MMP-2/TIMP-2 ratio may be of value in evaluating the prognosis in canine oronasal cavity tumors.

Maxillofacial rhabdomyosarcoma in the canine maxillofacial area.

Three dogs had a diagnosis of maxillofacial rhabdomyosarcoma. These dogs were treated with surgery and/or radiotherapy, and had poor clinical responses. The tumor tissues in all three cases were observed around the upper premolar teeth with ulcerative lesions and CT examinations in each case revealed extensive bony involvement into the maxilla. Two cases were subjected to surgical excision of the tissues, followed by an external radiation therapy. The other case was only treated with palliative radiation. Outcomes of the treatment of all the cases were quite poor because of the invasive and refractory nature of the tumor cells, leading to the local recurrence and lung metastasis early in the clinical course. All dogs died within two months of the first admission.

Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines.

Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.

Primary chondrosarcoma in the skull of a dog.

Chondrosarcoma of the skull is a rare primary malignant tumor that is slow-growing, but locally aggressive. A 5- year-old, golden retriever was presented to our hospital with a swelling in the left side of her head, and the swelling had slowly enlarged over the previous month. There were no significant changes on the neurological examination. A computed tomography scan revealed a large mass involving bone destruction and prominent matrix mineralization. T1-weighted magnetic resonance imaging showed a slightly low-signal intensity area and a T2-weighted image revealed marked, high-signal intensity. There was compression of the adjacent brain parenchyma. Histopathological examination confirmed the lesion to be a chondrosarcoma.

Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells.

This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs). ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs) and murine ADSCs (mADSCs) to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar ß-catenin and Akt phosphorylation between mADSCs and cADSCs. On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.