The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.     

Performance of a new carbon dioxide absorbent,Yabashi lime? as compared to conventional carbon dioxide absorbent during sevoflurane anesthesia in dogs

In the present study, we compare a new carbon dioxide (CO₂) absorbent, Yabashi lime^® with a conventional CO₂ absorbent, Sodasorb^® as a control CO₂ absorbent for Compound A (CA) and Carbon monoxide (CO) productions. Four dogs were anesthetized with sevoflurane. Each dog was anesthetized with four preparations, Yabashi lime^® with high or low-flow rate of oxygen and control CO₂ absorbent with high or low-flow rate. CA and CO concentrations in the anesthetic circuit, canister temperature and carbooxyhemoglobin (COHb) concentration in the blood were measured. Yabashi lime^® did not produce CA. Control CO₂ absorbent generated CA, and its concentration was significantly higher in low-flow rate than a high-flow rate. CO was generated only in low-flow rate groups, but there was no significance between Yabashi lime^® groups and control CO₂ absorbent groups. However, the CO concentration in the circuit could not be detected (≤5ppm), and no change was found in COHb level. Canister temperature was significantly higher in low-flow rate groups than high-flow rate groups. Furthermore, in low-flow rate groups, the lower layer of canister temperature in control CO₂ absorbent group was significantly higher than Yabashi lime^® group. CA and CO productions are thought to be related to the composition of CO₂ absorbent, flow rate and canister temperature. Though CO concentration is equal, it might be safer to use Yabashi lime^® with sevoflurane anesthesia in dogs than conventional CO₂ absorbent at the point of CA production.     

Itokawa dust particles: a direct link between S-type asteroids and ordinary chondrites

The Hayabusa spacecraft successfully recovered dust particles from the surface of near-Earth asteroid 25143 Itokawa. Synchrotron-radiation x-ray diffraction and transmission and scanning electron microscope analyses indicate that the mineralogy and mineral chemistry of the Itokawa dust particles are identical to those of thermally metamorphosed LL chondrites, consistent with spectroscopic observations made from Earth and by the Hayabusa spacecraft. Our results directly demonstrate that ordinary chondrites, the most abundant meteorites found on Earth, come from S-type asteroids. Mineral chemistry indicates that the majority of regolith surface particles suffered long-term thermal annealing and subsequent impact shock, suggesting that Itokawa is an asteroid made of reassembled pieces of the interior portions of a once larger asteroid.     

A hydro-environmental watershed model improved in canal-aquifer water exchange process

Long-term simulation using the distributed hydro-environmental watershed model is efficacious for assessing irrigation impacts on hydrological cycle in detail and for implementing watershed management successfully. In this article, the previously developed hydro-environmental watershed model (HEWM-1) is improved in the water exchange process caused by surface water-groundwater interaction via drainage canals and/or underdrains. The time-varying stream flow in canals is described by the complete one-dimensional shallow water equations in a newly introduced submodel, the open channel flow submodel. This submodel coordinates with the other submodels: the tank, soil moisture and groundwater flow submodels which are interlinked in a cascade manner. The improved model (HEWM-2) is applied to an agricultural watershed covering an area from an alluvial fan onto a nearly level alluvial plain, to be validated. The simulation by HEWM-2 is informative for identifying whether any drainage canal is gaining or losing water in relation to groundwater level. It could thus provide useful information for conserving a complex network of drainage canals which also functions as a passage for aquatic animals like fishes.     

A stochastic model for behaviour of fish ascending an agricultural drainage system

Adults of Carassius auratus grandoculis ascend from Lake Biwa, Japan, into riparian paddy fields via agricultural drainage systems for the purpose of spawning. However, land improvement works conducted in the second half of the twentieth century have brought about obstacles for passage of the fish, and several restoration projects to recover the migratory environment are being attempted. A continuous Markov process model is presented to scientifically support such a project, defining an ascending probability that quantitatively assesses the ability of an agricultural drainage system with hydraulic structures for the ascent of the fish. Computational methods are developed to numerically obtain flow fields of the agricultural drainage system and then the ascending probability in the entire spatio-temporal domain. The values of model parameters are deductively inferred, referring to results of field observations. From the computational results of the ascending probability in the agricultural drainage system, effects of hydraulic structures allowing ascent of the fish are clarified.     

Herpes Simplex Virus Type 1 Infection in Two Pet Marmosets in Japan

An 8-month-old common marmoset (Callithrix jacchus) was presented with tic-like symptoms, and a 2-year-old pigmy marmoset (Callithrix pygmaea) was presented with dyspnea and hypersalivation. Both monkeys died within a few days, and necropsies were performed. Histopathological examinations revealed ulcerative stomatitis with epithelial cell swelling and eosinophilic intranuclear inclusion bodies in the oral epithelium of both cases. In the central and peripheral nervous systems, neuronal cell degeneration with intranuclear inclusion bodies was observed. Immunohistochemical examination using anti-herpes simplex virus type 1 antibody revealed virus antigens in both cases. Both animals had been kept as pets with limited exposure to the ambient environment except via their owners. Therefore, herpes simplex virus type-1 was probably acquired from close contact with their owners.     

Bending resistance of repaired column members and shear resistance of opening frames with repaired columns of conventional Japanese wooden houses

When it is necessary to repair conventional Japanese wooden houses, the decayed lower parts of columns should be replaced with new wood material. The bending resistance of columns repaired by four methods and the shear resistance of opening frames with those repaired columns were investigated in this study. Bending tests of the repaired columns showed differences in initial bending stiffness and maximum bending moment related to the repair methods and loading direction. Racking tests were conducted on door opening frames with conventional door head members or upper partial walls sheathed with 12-mmthick plywood. The conventional frame specimens broke at door head-column joints with no obvious bending deformation of the columns, resulting in little difference in load-shear deformation curves among the repair methods. The columns of plywood-sheathed specimens, on the other hand, clearly were bent after the nails at the plywood-to-wood frame joints started to pull off. The load-shear deformation curves of the plywood-sheathed specimens did not vary regardless of the repair methods when shear deformations were small but were affected by repair methods as shear deformation increased.     

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O2 compared to 5% O2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O2 tension, whereas IVD101 supported blastocyst formation only under low O2 levels but enhanced the proliferation of ICM cells.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

Vascular distribution in ruptured canine cranial cruciate ligament

Objective: To (1) determine the microanatomic vascular distribution in ruptured canine cranial cruciate ligaments (CCL) using specific vascular immunohistochemical techniques, and (2) compare vessel density between ruptured and intact canine CCL and between different areas of interest in ruptured CCL using histomorphometric analysis. Study Design: In vitro study. Animals: Dogs (n=41) admitted for surgical treatment of ruptured CCL and 19 dogs euthanatized for nonorthopedic conditions. Methods: Diseased (variable CCL rupture) and intact (normal control) CCL were processed for immunohistochemical staining specific to vessels (factor VIII, laminin). Mean vascular density was assessed and compared in areas of interest (torn end versus remaining core regions of CCL, proximal femoral versus distal tibial core CCL regions). Results: Ruptured CCL was more vascular than intact CCL; however there was no difference in vascular density between the torn end and the remaining core area of the ruptured CCL. Ruptured CCL was vascularized to a greater degree at the proximal portion than the distal portion of the CCL. Partially ruptured CCLs had a higher vessel density than completely ruptured CCLs. Conclusions: Vascular density is increased in diseased CCL compared with intact CCL. It remains to be determined whether this finding is associated with the cause of CCL rupture or is a result of CCL degeneration and rupture.