Development of a Medium-term Animal Model Using gpt Delta Rats to Evaluate Chemical Carcinogenicity and Genotoxicity

In this study, the potential for development of an animal model (GPG46) capable of rapidly detecting chemical carcinogenicity and the underlying mechanisms of action were examined in gpt delta rats using a reporter gene assay to detect mutations and a medium-term rat liver bioassay to detect tumor promotion. The tentative protocol for the GPG46 model was developed based on the results of dose-response exposure to diethylnitrosamine (DEN) and treatment with phenobarbital over time following DEN administration. Briefly, gpt delta rats were exposed to various chemicals for 4 weeks, followed by a partial hepatectomy (PH) to collect samples for an in vivo mutation assay. The mutant frequencies (MFs) of the reporter genes were examined as an indication of tumor initiation. A single intraperitoneal (ip) injection of 10 mg/kg DEN was administered to rats 18 h after the PH to initiate hepatocytes. Tumor-promoting activity was evaluated based on the development of glutathione S-transferase placental form (GST-P)-positive foci at week 10. The genotoxic carcinogens 2-acetylaminofluorene (2-AAF), 2-amino-3-methylimidazo [4,5-f] quinolone (IQ) and safrole (SF), the non-genotoxic carcinogens piperonyl butoxide (PBO) and phenytoin (PHE), the non-carcinogen acetaminophen (APAP) and the genotoxic non-hepatocarcinogen aristolochic acid (AA) were tested to validate the GPG46 model. The validation results indicate that the GPG46 model could be a powerful tool in understanding chemical carcinogenesis and provide valuable information regarding human risk hazards.     

A simple and rapid immunocytochemical technique for detection of cytokeratin, vimentin, and S-100 protein in veterinary diagnostic cytology

The objective of this study was to establish a simple and rapid immunocytochemical technique that can be used in veterinary diagnostic cytology. Air-dried impression smears were collected from canine tumors. Samples of epithelial tumors, mesenchymal tumors, and malignant peripheral nerve sheath tumors and melanomas were used for detection of cytokeratin, vimentin, and S-100 protein, respectively. The labeled streptavidin-biotin system was used in the present study. Optimal fixation was determined using standard immunocytochemical procedures, and acetone fixation was found to be the most effective. Optimal concentrations of primary and secondary antibodies were determined at a preset 5-min incubation. Omission of H(2)O(2) treatment, shortening the time for blocking and labeled-streptavidin incubation, and simplifying washing did not decrease immunopositive intensities or enhance false-positive reactions. The described rapid protocol requires approximately 45min without the use of any special equipment.     

Cryopreservation of pig spermatozoa using carboxylated poly-L-lysine as cryoprotectant

In this study, we cryopreserved pig spermatozoa using carboxylated poly-L-lysine (CPLL) as the cryoprotectant to determine its efficacy. Pig spermatozoa were placed in a freezing extender containing 3% (v/v) glycerol and different CPLL concentrations. The motility indices of the spermatozoa cryopreserved with 0.25% (v/v) CPLL at 6 (59.3), 9 (53.7), and 12 (26.2) h after thawing were significantly higher (P < 0.01 or P < 0.05) than those of the spermatozoa cryopreserved without CPLL (53.7, 40.1, and 17.5 at 6, 9, and 12 h after thawing, respectively). The concentration of CPLL in the freezing extender did not affect the ability of frozen-thawed spermatozoa to fertilize oocytes in vitro. However, the blastocyst formation rate of embryos derived from spermatozoa cryopreserved with 0.25% CPLL (24.6%) was significantly higher (P < 0.01) than that of embryos derived from spermatozoa cryopreserved without CPLL (11.2%). The conception rate of the sows inseminated with spermatozoa cryopreserved with 0.25% CPLL (72.2%) was not significantly different from that of the sows inseminated with spermatozoa stored at 17°C (81.3%). However, the mean number of total piglets born to the former (10.0) was significantly lower (P < 0.05) than that of total piglets born to the latter (13.4). The results showed that CPLL in the freezing extender maintained the motility of frozen-thawed pig spermatozoa and improved the in vitro development of embryos produced by in vitro fertilization. In addition, we have demonstrated that piglets could be obtained with artificial insemination using spermatozoa cryopreserved with CPLL.     

Importance of albumen content in whole-body protein synthesis of the chicken embryo during incubation.

The importance of egg albumen content in whole-body protein synthesis was investigated in developing chicken embryos by using lines genetically selected for high and low albumen contents and by removing albumen from eggs before incubation. 2. Whole-body protein synthesis was estimated by injecting L-[15N]-phenylalanine intravenously on day 12 of incubation. 3. Embryos from high albumen eggs had higher whole-body protein synthesis rates than those from low albumen eggs. 4. Whole-body protein synthesis was reduced by the removal of albumen from eggs before incubation. 5. It was concluded that albumen content per se was of crucial importance in regulating whole-body protein synthesis in chicken embryos during incubation.     

Axial correction of pes varus by transverse-opening wedge osteotomy and T-plate fixation with beta-tricalcium phosphate (beta-TCP) transplantation in dachshunds

Axial correction was performed surgically in two miniature dachshunds presenting with lateral patellar dislocation and limping caused by pes varus. Pes varus had resulted from asymmetric closure of the physis of the distal tibia. Prior to surgery, osteotomy was simulated by measuring X-ray films to determine the distance required for the wedge opening. Transverse-opening wedge osteotomy was performed on the medial side of the distal tibia, and beta-tricalcium phosphate (beta-TCP) was inserted in a wedge shape into the area created by the cuneiform osteotomy. Finally, the tibia was fixed by a veterinary 1.5/2.0-mm T-plate. Both dogs were able to walk a few days after surgery, and the lateral dislocation of the patella normalized almost completely in about one month. At two months, X-ray films showed that the implant had remained in position without any dislocation, and the beta-TCP had fused with the surrounding bone.     

Interpreting gelatinase activity in tumor tissue and serum as a prognostic marker of naturally developing canine tumors

To evaluate the clinical usefulness of tissue and serum gelatinase activity as a prognostic marker of canine tumors, tissue samples from 60 tumors and corresponding serum samples from the same animals were collected at the time of biopsy and surgery. On the basis of histopathology and clinical aggressiveness of metastasis and recurrence (MR), the cases were divided into 6 categories: non-inflammatory (Inf(-)) and inflammatory (Inf(+)) benign, and the Inf(-) MR(-), Inf(-) MR(+), Inf(+) MR(-), and Inf(+) MR(+) malignant. Gelatinase activity was determined semi-quantitatively using gelatin zymogram with a gelatinase standard from cultured canine peripheral blood mononuclear cells stimulated with lipopolysaccharide. No significant difference in gelatinase activities in tissue extracts was evident between the benign and malignant tumors. Inf(+) benign tumors, as well as Inf(-) MR(+), Inf(+) MR(-) and Inf(+) MR(+) malignant tumors, showed significantly higher tissue gelatinase activity than Inf(-) benign. The tissue activity in Inf(-) MR(-) malignant was significantly lower than in Inf(+) MR(-) and Inf(+) MR(+) malignant. The serum activity was significantly higher in the malignant cases than in the control and the benign. Inf(-) MR(+), Inf(+) MR(-) and Inf(+) MR(+) malignant tumors induced significantly higher gelatinase activity in serum than Inf(-) benign tumors. Gelatinase activity in serum was positively correlated with that in tumor extracts. Increased gelatinase in tumor tissue and serum may be correlated with inflammation as well as tumor aggressiveness, and thus should be used in combination with histopathology for predicting tumor metastasis or recurrence.     

Seasonal influence on testicular function of male raccoons, Procyon lotor

In order to clarify the breeding capability of male raccoons in Japan, the testes of raccoons, a nuisance animal, collected in Kanagawa Prefecture and Hokkaido were histologically inspected. Furthermore, testosterone concentrations in their blood were measured. The testosterone concentrations increased in winter and the diameter of seminiferous tubules and the spermatogenetic score decreased in summer for the animals captured in Kanagawa Prefecture. For the animals captured in Hokkaido, the diameter of seminiferous tubules did not change and the decrease of the spermatogenetic score in summer was slight. As the above results show, there is a breeding season in male raccoons in Japan, and the reduction of testicular function in summer was greater in animals captured in Kanagawa Prefecture than in animals captured in Hokkaido.     

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.