Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

Proliferation of equine bone marrow-derived mesenchymal stem cells in gelatin/β-tricalcium phosphate sponges

A three dimensional scaffold is essential in mesenchymal stem cells (MSCs) delivery in cell-based therapy for facilitating cell adherence, migration, proliferation, and differentiation. The objectives of this study were to evaluate the possibility of β-tricalcium phosphate incorporated gelatin sponges (Gelatin/β-TCP sponge) as scaffolds for equine MSCs and to examine the effects of seeding density and seeding method on the proliferation of equine MSCs in the Gelatin/β-TCP sponges. Mononuclear cells and MSCs isolated from bone marrow were seeded into Gelatin/β-TCP sponges at different densities by different seeding methods-static or agitated methods. Proliferation of the MSCs in Gelatin/β-TCP was assessed using the Cell Counting Kit-8 assay and histological examination. Distribution and proliferation of MSCs in the Gelatin/β-TCP sponge were observed, and the Gelatin/β-TCP sponge supported limited growth when seeded at high density. We also found that the agitated seeding method enhanced the proliferation of MSCs. This study demonstrated the suitability of Gelatin/β-TCP sponges for the proliferation and maintenance of equine MSCs. These results contribute to the application of MSC-seeded Gelatin/β-TCP sponges in equine medicine.     

Seroepidemiological survey of sheep flocks from Northern Japan for Mycoplasma ovipneumoniae and Mycoplasma agalactiae

Sheep flocks from Hokkaido, Iwate and Aomori, three northern prefectures of Japan, were screened for antibodies to Mycoplasma ovipneumoniae and Mycoplasma agalactiae by ELISA. Sixty four animals out of 246 (26%) were seropositive to M. ovipneumoniae, with positive results obtained from all three prefectures. None of the sera tested were serologically positive to M. agalactiae.     

Effects of feeding early‐harvested orchardgrass–perennial ryegrass mixed silage instead of heading stage harvested timothy silage on digestion and milk production in dairy cows

We evaluated the effects of replacement of heading stage harvested timothy silage with early‐harvested orchardgrass–perennial ryegrass mixed (OP) silage while maintaining or reducing concentrate input on dry matter intake (DMI), milk production, nutrient digestibility, and N balance in dairy cows. Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design with three dietary treatments: TYL, a diet containing timothy silage where forage‐to‐concentrate ratio (FC) was 50:50; OPL, a diet containing OP silage where FC ratio was 50:50; and OPH, a diet containing OP silage where FC ratio was 60:40. We observed that an equal replacement of timothy with OP silage increased DMI, milk yield, milk protein production, and nutrient digestibility but decreased milk fat content (TYL versus OPL). We observed that replacing timothy with OP silage while reducing concentrate input increased milk fat and protein yield, nutrient digestibility, and feed efficiency and reduced urinary N loss with no effect on DMI or milk fat content (TYL versus OPH). These results show that replacing timothy with OP silage can be a good approach to improve milk production, feed efficiency, and N utilization and reduce concentrate input. However, milk fat depression should be considered when an equal substitution is performed.     

Establishment of a syngeneic orthotopic model of prostate cancer in immunocompetent rats

We previously established 3 cell lines (PLS10, PLS20 and PLS30) from a chemically-induced prostate carcinoma in F344 rats, and demonstrated high potential for metastasis in nude mice. In the present study, we investigated the feasibility of establishing an orthotopic model using the 3 rat prostate cancer cell lines in immunocompetent rats with the aim of resolving species-mismatch problems and defects of immune systems. The PLS10, PLS20 and PLS30 cell lines were injected into the ventral prostates of 6-week-old rats, which were then sacrificed at experimental weeks 4 and 8. Tumor mass formation was found in rats with PLS10, but not in those with PLS20 or PLS30. Additionally, metastatic carcinomas could be detected in lymph nodes and lungs of PLS10-inoculated rats. Genetic analysis demonstrated K-ras gene mutations in PLS10 and PLS20, but not in PLS30 cells. There were no mutations in p53 and KLF6. In conclusion, we established a syngeneic orthotopic model for prostate cancer in immunocompetent rats simulating human castration-resistant prostate cancer (CRPC), which should prove useful for development and validation of therapeutic agents, especially with immunotherapy.     

ErbB2 copy number gain is associated with adverse outcome in canine mammary carcinoma

Copy number gain (CNG) and/or protein overexpression of ErbB2 have been observed in human breast cancer patients and are associated with poor prognosis. Similarly, ErbB2 overexpression has also been observed in canine mammary carcinoma; however, data on ErbB2 copy number is limited. The purposes of this study were to evaluate ErbB2 copy number in dogs with mammary carcinoma and to investigate associations of ErbB2 CNG with ErbB2 expression, histological and clinical characteristics, and survival. DNA samples were isolated from 59 formalin-fixed paraffin-embedded canine mammary gland tissues (34 carcinoma, 14 adenoma, and 11 normal). Using a digital PCR assay, the ErbB2 copy number in these samples was determined as compared to a reference gene on canine chromosome 8. ErbB2 CNG was detected in 14/34 (41%) carcinomas and 2/14 (14%) adenomas. ErbB2 overexpression was observed in 3/34 (9%) carcinomas but not in adenomas. Neither ErbB2 CNG nor ErbB2 overexpression were detected in the normal controls. There was no significant association of the ErbB2 CNG with histological and clinical characteristics such as age, neutered status, histological grade, tumor size, lymph node involvement, distant metastasis, and clinical stage in the dogs with mammary carcinoma. The presence of ErbB2 CNG, but not ErbB2 overexpression, was significantly related to the shorter overall survival. These findings suggest that ErbB2 CNG is a prognostic factor in dogs with mammary carcinoma.     

Evaluating the use of piezo manipulator,laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B < 700 µm and C ≥ 700 µm). Six embryos 233–1360 µm in diameter were punctured via piezo manipulator and/or laser pulse before cryopreservation. All embryos were cryopreserved on a Cryotop®. Frozen-thawed embryos were cultured for 3h and transferred to the recipient mares. After one week, pregnancy was diagnosed by ultrasonography. Two of six embryos resulted in a positive pregnancy, the result of pregnancy in group A and B was positive, but in group C was negative, and further investigation is necessary for ≥700 µm embryos. The results showed laser-assisted puncture could be helpful to extract blastocoel fluid and replace it with cryoprotectant. This is the first positive pregnancy report in laser puncture-assisted frozen-thawed equine embryo (>300 µm). However, more research is required to find the best method for embryos ≥700 µm.     

Regulative actions of dietary soy isoflavone on biological antioxidative system and lipid metabolism in rats

Male Sprague-Dawley rats, 4 weeks of age, were fed purified diets either with or without 0.2% soy isoflavones rich powder for 5 weeks to elucidate their direct functions such as antioxidative action and regulation of lipid metabolism. Dietary soy isoflavones decreased serum lipid peroxide level in rats. Levels of liver and serum alpha-tocopherol were higher in the rats fed isoflavone than in those fed isoflavones-free diet. Thus, dietary soy isoflavones exhibited mild antioxidative function in this animal experiment. Isoflavone metabolites from diet may act as scavengers of reactive oxygen species. Dietary soy isoflavones lowered hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, although liver cholesterol level was not modulated. However, the levels of serum cholesterol and triglyceride decreased by consumption of soy isoflavones. Therefore, dietary soy isoflavones may exhibit hypocholesterolemic and hypolipidemic functions. Moreover, dietary soy isoflavones lowered hepatic Delta6 desaturase activity. Reflecting this observation, Delta6 desaturation indices ((18:2(n = 6) + 18:3(n = 6))/20:4(n = 6)) of tissue lipids tended to be lower in rats fed isoflavones than in those fed isoflavones-free diet. This action may contribute to the prevention of inflammatory response by imbalance of eicosanoids. These observations suggest that the positive intake of soy isoflavones may reduce the risk of some cardiovasucular diseases through their radical scavenging function and hypocholesterolemic action.     

Extraction of Soil Organic Nitrogen by Organic Acids and Role in Mineralization of Nitrogen in Soil

The solubilization of organic nitrogen (Norg) in soil was examined by using organic acids often present in root exudates. The organic acids tested were classified into two groups depending on their solubilizing ability for Norg in a soil sample. Oxalate, malonate, tartarate, citrate and malate could dissolve Norg by increasing their concentrations, while succinate and glutarate could not dissolve Norg even when their concentrations were increased. The amount of organic N extracted with oxalate, malonate, tartarate, citrate and malate showed a strong correlation with the sum of the amounts of Al and Fe in the extracts. Therefore, it was assumed that the solubilizing ability may be attributed to the structure of these organic acids that can form stable chelate compounds with Al and Fe (oxalate, malonate, tartarate, citrate and malate). Furthermore, the amount of 20 mM cit-rate-extractable Norg (CEON), which was the highest among the organic acids tested, showed a strong correlation with both that of 67 mM phosphate buffer, which is now being widely used as an index for estimating soil N availability in Japan, and N mineralized using an incubation method in 46 soil samples belonging to different soil types. These results suggested that CEON might be an important constituent of mineralizable Norg in soil. Therefore, organic acids secreted by crops play a role in the solubilization of available Norg surrounding the crop rhizosphere.     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.