Effects of trichostatin A on in vitro development and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells

The present study was carried out to examine the effects of post‐activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo‐β‐galactosidase C gene (removal of the α‐galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate‐labelled BS‐I‐B₄ isolectin, the intensity of fluorescence observed on cell‐surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non‐transgenic SCNT blastocyst. However, the reduction of α‐Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post‐activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.     

Successful suppression of endogenous α-1,3-galactosyltransferase expression by RNA interference in pig embryos generated in vitro

RNA interference (RNAi) technology using small interfering RNAs (siRNA) has been widely used as a powerful tool to knock down gene expression in various organisms. In pig preimplantation embryos, no attempt to suppress the target gene expression with such technology has been made. The purpose of this study is to demonstrate that the RNAi technology is useful for suppression of endogenous target gene expression at an early stage of development in pigs. Alpha-1,3-Galactosyltransferase (α-GalT) is an enzyme that creates the Galα1-3Gal (α-Gal) epitope on the cell surface in some mammalian species, and removal of the epitope is considered to be a prerequisite for pig-to-human xenotransplantation. We decided to suppress the endogenous α-GalT mRNA expression in pig early embryos, since reduction of α-GalT synthesis is easily monitored by cytochemical staining with Bandeiraea simplicifolia isolectin-B(4), a lectin that specifically binds to the α-Gal epitope, and by RT-PCR analysis. Cytoplasmic microinjection of double-stranded RNA and pronuclear injection of an siRNA expression vector into the embryos generated in vitro resulted in a significant reduction in expression of the α-GalT gene and α-Gal epitope in blastocysts, at which stage the α-Gal epitope is abundantly expressed. Somatic cell nuclear transfer of embryonic fibroblasts stably transfected with an siRNA expression vector also led to a significant reduction in the level of α-GalT mRNA synthesis together with decreased amounts of the α-Gal epitope at the blastocyst stage. These results indicate that the RNAi technology is useful for efficient suppression of a target gene expression during embryogenesis in pigs and suggest the possibility of production of siRNA-expressing pigs for use in xenotransplantation.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

Porencephaly in dogs and cats: relationships between magnetic resonance imaging (MRI) features and hippocampal atrophy

Porencephaly is the congenital cerebral defect and a rare malformation and described few MRI reports in veterinary medicine. MRI features of porencephaly are recognized the coexistence with the unilateral/bilateral hippocampal atrophy, caused by the seizure symptoms in human medicine. We studied 2 dogs and 1 cat with congenital porencephaly to characterize the clinical signs and MRI, and to discuss the associated MRI with hippocampal atrophy. The main clinical sign was the seizure symptoms, and all had hippocampal atrophy at the lesion side or the larger defect side. There is association between hippocampal atrophy or the cyst volume and the severe of clinical signs, and it is suggested that porencephaly coexists with hippocampal atrophy as well as humans in this study.     

Preincubation with green tea polyphenol extract is beneficial for attenuating sperm injury caused by freezing‐thawing in swine

Polyphenols (PFs) extracted from green tea, known to be potent anti‐oxidants, have been reported to be effective in increasing the motility and viability of mammalian sperm, preserved in a liquid form. Therefore, we tested whether PFs might also be effective for maintaining the integrity of frozen‐thawed boar spermatozoa. Ejaculates, collected from Clawn miniature pigs, were diluted in a semen extender containing various amounts of PFs (0, 0.01, 0.05, 0.1 and 0.2% w/v) and then stored at 15°C overnight. The semen samples were processed, using the straw freezing procedure, and then frozen in liquid nitrogen. After rapid thawing at 40°C, the spermatozoa were subjected to several assays to evaluate semen quality. Spermatozoa frozen in a medium containing 0.01% w/v PFs exhibited significantly (P < 0.05) higher degrees of post‐thawed viability and acrosomal integrity than those stored in the absence of PFs. However, no change in the mitochondrial activity was noted between the two groups. The inclusion of 0.01% PFs in the semen extender was significantly (P < 0.05) effective in increasing both the rates of monospermic oocyte formation and of blastocyst formation. These findings indicate that preincubation with the semen extender, containing 0.01% PFs prior to freezing, exerts a protective effect on boar sperm by preventing injuries associated with freezing‐thawing.     

A new rolling culture‐based in vitro fertilization system capable of reducing polyspermy in porcine oocytes

The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 10⁵ sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration.     

The effects of gel pad thickness on the evaluation of skin structures using ultrasonography in normal dogs

Gel pads are commonly used in skin ultrasonography; however, the effects of their thickness are unknown. This study investigated the effects of pad thickness on measurements of skin thickness in 10 beagle dogs. Sonograms to measure neck skin thickness were captured without pads and using pads with thicknesses of 3, 5, 10, and 20 mm. Without pads, acoustic shading was observed due to air bubbles in the coupling gel. With 20-mm pads, echogenic artifacts were observed on the skin surface. Entry echo with 20-mm pads was significantly higher than with 3-mm pads. This suggests that visibility of the skin structure could be affected when a gel pad is not used or when a thick gel pad is selected.     

Imatinib mesylate treatment in a dog with gastrointestinal stromal tumors with a c-kit mutation

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, Asn⁵⁰⁸Ile), 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.     

A comparison of the immunological effects of propofol and isoflurane for maintenance of anesthesia in healthy dogs

Most anesthetics have an immuno-suppressive effect on cellular and neurohumoral immunity, and research shows that total intravenous anesthesia (TIVA) with propofol has a greater immuno-protective effect than inhalational anesthesia in human medicine. However, in veterinary clinics, these effects remain ambiguous. To clarify the details, we focused on propofol and isoflurane, investigating clinical blood hematology and immunological profiles drawn from healthy dogs under and after two anesthesia techniques. Twelve healthy adult beagles were included in this study, randomly assigned to the propofol anesthesia group (group P: n=6) or the isoflurane anesthesia group (group I: n=6). In both groups, the number of lymphocytes in peripheral blood decreased after 2 hr of anesthesia (2 hr), but group P showed significantly less decrease than group I. For T-lymphocyte subsets examined by flowcytometry, the ratio of CD3+, CD4+ and CD8+ lymphocytes in the peripheral blood mononuclear cell (PBMC) of group P at 2 hr also exhibited a high level compared to group I. Moreover, for mRNA expression of cytokines measured by real-time PCR, the IL2 (pro-inflammatory cytokine) of group P showed no decrease like group I. The IL10 (anti-inflammatory cytokine) of group P also showed no increase like group I, while both cytokines maintained nearly the same level until 2 hr. These results suggest that, compared to propofol, isoflurane had more strongly immuno-suppression caused by anesthesia, and propofol itself might have some immuno-protective effects. Thus, TIVA with propofol might benefit immunological support in the perioperative period of dogs.     

Mediastinal basaloid carcinoma arising from thymic cysts in two dogs

Mediastinal masses in dogs were diagnosed as basaloid carcinoma associated with multiple thymic cysts (MTCs). The masses were composed of MTCs and proliferating intracystic neoplastic basaloid cells, which immunohistochemically diffusely expressed p63 and cytokeratin 19. A gradual transition from the basal cell layers lining the cysts walls to the neoplastic cells was seen, and it was indicated that the neoplastic cells had originated from the basal cell layers of the cysts. To the best of our knowledge, this is the first report of basaloid carcinoma occurring in the mediastinal cavity in dogs. Although these tumors were demonstrated to be rare origins, basaloid carcinoma should be included in the differential diagnoses for canine mediastinal tumors.