Spontaneous Accumulation of Globotriaosylceramide (Gb3) in Proximal Renal Tubules in an ICR Mouse

This report describes spontaneous cytoplasmic vacuolation in the proximal renal tubules of a 7-week-old male ICR [Crlj:CD1(ICR)] mouse. The contents of vacuoles were positively stained with periodic acid-Schiff (PAS) and Sudan black, and the membranes were positive on immunohistochemical staining for lysosomal-associated membrane protein-2 (LAMP-2), a marker of lysosomal membrane. Electron microscopy revealed electron-dense lamellar bodies in the proximal tubular epithelial cells. These histopathological features are similar to those in α-galactosidase A-deficient mice, in which globotriaosylceramide (Gb3), a glycosphingolipid, accumulates in lysosomes. When we performed immunohistochemical staining for Gb3, the contents of vacuoles were positively stained. From these results, spontaneous cytoplasmic vacuolation in the proximal renal tubules in the mouse was identified as lysosomal accumulation of Gb3.     

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.     

CT and MRI imaging diagnosis of epidural idiopathic sterile pyogranulomatous inflammation in a dog spinal canal

A 12-year-old neutered male shih tzu developed progressive pelvic limb paraparesis. Computed tomography showed a radiolucent mass lesion in the spinal canal at the left side of the 11th thoracic vertebra. The mass was not enhanced by intravenous contrast medium injection. It was hyperintense on both T1- and T2-weighted magnetic resonance images. The signal intensity of the mass was decreased with a fat suppression technique, indicating a fatty origin. After removal of the mass via T11-T12 hemilaminectomy, chronic panniculitis was confirmed by histopathological examination. This case demonstrates the utility of computed tomography and magnetic resonance imaging for the diagnosis of spinal canal pyogranulomatous inflammation.     

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.     

Amplification of the c-yes oncogene in canine mammary tumors

Genomic DNAs of 14 mammary tumors were analyzed by Southern blot hybridization using a human c-yes-1 oncogene probe. The amplification was successful in half of the cases (7 adenocarcinomas). The degree of amplification was approximately 4-fold, and a high proportion was seen in malignant tumors. In addition, DNA polymorphism was detected in two adenocarcinomas.     

Comparison of noninvasive cardiac output measurements by transthoracic bioimpedance, partial carbon dioxide rebreathing, and transesophageal echocardiography with the thermodilution technique in beagle dogs

Most methods for determining cardiac output (CO) have limited application in clinical practice due to the invasive techniques required. This study compared the thermodilution technique (TDCO) with three noninvasive methods for determining CO in anesthetized dogs: transthoracic bioimpedance (BICO), partial CO₂ rebreathing (NICO), and transesophageal echocardiography (TEECO). TDCO was compared to BICO, NICO, and TEECO in six adult sevoflurane anesthetized beagle dogs (9.1–13.0 kg). All dogs were administered midazolam [0.3 mg kg^?1, intravenously (IV)] and butorphanol (0.1 mg kg^?1 IV), followed by ketamine (5.0 mg kg^–1 IV) and sevoflurane in nitrous oxide (1 L minute^–1) and oxygen (1 L minute^–1) and mechanically ventilated. Dogs were maintained at 2.2% end‐tidal sevoflurane (ETsev) concentration for instrumentation and baseline measurements. Low (5.0% ETsev), intermediate (3.3% ETsev), and high cardiac output values were achieved by varying the end‐tidal sevoflurane concentration and the administration of dobutamine (3–10 g kg^–1 minute^–1 and 2.2% ETsev). A minimum of thirty data sets was obtained for each comparison. The correlation coefficients when compared to TDCO were 0.684 for BICO (p < 0.0001), 0.883 for NICO (p < 0.0001), and 0.991 for TEECO (p < 0.0001). Cardiac output values ranged 50–444 mL kg^–1 minute^–1 for TDCO, 100–253 mL kg^–1 minute^–1 for BICO, 64–214 mL kg^–1 minute^–1 for NICO, and 52–401 mL kg^–1 minute^–1 for TEECO. The differences when compared to TDCO ranged – 62–235 mL kg^?1minute^?1 for BICO, 18–220 mL kg^?1 minute^?1 for NICO, and – 35–32 mL kg^–1 minute^–1 for TEECO. Differences were maximum at the highest CO in BICO and NICO. In conclusion, this study demonstrated that BICO and NICO underestimate CO in sevoflurane anesthetized dogs. TEECO is a viable noninvasive method for determining CO in sevoflurane anesthetized dogs.     

Prediction of crude fat content of longissimus muscle of beef using the ratio of fat area calculated from computer image analysis: comparison of regression equations for prediction using different input devices at different stations

Crude fat content of longissimus (ribeye) muscle of beef cattle was predicted from a ratio of fat area (RFA) to area of ribeye muscle calculated from computer image analysis (CIA). Cross sections of 64 ribeyes taken from the 6-7th rib from cattle at experiment station A and cross sections of 94 ribeyes taken from the 6-7th rib from cattle at Experiment Station B were used in this study. Slices (1 to 1.5 cm in thickness) of just the Longissimus dorsi were homogenized and sampled for chemical estimation of crude fat content using petroleum ether. Crude fat content as determined from chemical analysis was used as the true estimate of fat content. A CCD (charge-coupled device) camera was used as the input device at Experiment Station A, and a single-lens reflex camera was used at Experiment Station B to photograph ribeyes for CIA. The contour comparison method, which assigns a threshold value for each marbling particle, was used to obtain accurate binarization in this study. Minimum and maximum of chemical measurements of crude fat were 2.1 and 39.8%, and for CIA calculation of the RFA were 6.1 and 56.8%, respectively. This range covered almost the complete range of the beef marbling standard used in carcass grading in Japan. The equation for the regression of the crude fat content (Y) on RFA (X) calculated from CIA for all of the data was Y = .793X-3.04 with r2 = .96. Regression equations for prediction of crude fat percentage from RFA taking into consideration the effect of experiment station were Y = .741X-2.22 with r2 = .91 for Experiment Station A, and Y = .782X-2.54 with r2 = .91 for Experiment Station B. Analysis of covariance showed that the effects of experiment stations on intercepts and slopes were not significant (P > .10). The ranges of differences between actual and predicted crude fat content from the prediction equation that was calculated without consideration of the effect of station were -6.4 to 4.0%. CIA of cross sections of the ribeye muscle seems to have potential for prediction of crude fat content.     

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.