Normal DNA methylation status in sperm from a somatic cell cloned bull and their fertilized embryos

Epigenetic reprogramming confers totipotency even during somatic cell nuclear transfer (SCNT), which has been used to clone various animal species. However, as even apparently healthy cloned animals sometimes have aberrant epigenetic status, the harmful effects of these defects could be passed onto their offspring. This is one of the biggest obstacles for the application of cloned animals for livestock production. Here, we investigated the DNA methylation status of four developmentally regulated genes (PEG3, XIST, OCT4, and NANOG) in sperms from a cloned and a non‐cloned bull, and blastocysts obtained by in vitro fertilization using those sperms and SCNT. We found no differences in the methylation status of the above genes between cloned and non‐cloned bull sperms. Moreover, the methylation status was also similar in blastocysts obtained with cloned and non‐cloned bull sperms. In contrast, the methylation status was compromised in the SCNT blastocysts. These results indicate that sperm from cloned bulls would be adequately reprogrammed during spermatogenesis and, thus, could be used to produce epigenetically normal embryos. This study highlights the normality of cloned bull offspring and supports the application of cloned cattle for calf production.     

Malaria in cynomolgus monkeys used in toxicity studies in Japan

Plasmodium spp. protozoa cause malaria and are known to infect humans and a variety of animal species including macaque monkeys. Here we report both our experience with malaria recrudescence in cynomolgus monkeys (Macaca fascicularis) in a toxicity study and the results of a survey on Plasmodium infection in cynomolgus monkeys imported to Japan for laboratory use. A cynomolgus monkey from the toxicity study presented with severe anemia and Plasmodium protozoa in erythrocytes on a thin blood smear and was subsequently diagnosed with symptomatic malaria. In this animal, congestion and accumulation of hemozoin (malaria pigment) in macrophages were noted in the enlarged and darkly discolored spleen. As a follow-up for the experience, spleen sections from 800 cynomolgus monkeys in toxicity studies conducted between 2003 and 2013 were retrospectively examined for hemozoin deposition as a marker of Plasmodium infection. The origin of the animals included Cambodia, China, Indonesia, and Vietnam. Hemozoin deposition was confirmed in 44% of all examined monkeys. Monkeys from Indonesia showed the highest incidence of hemozoin deposition (approx. 80%). A high prevalence of Plasmodium infection in laboratory monkeys was also confirmed with polymerase chain reaction (PCR) by using Plasmodium genus-specific primers. Although Japan is not a country with endemic malaria, it is important to be aware of the prevalence and potential impact of background infection with Plasmodium spp. and recrudescence of symptomatic malaria in imported laboratory monkeys on pharmaceutical toxicity studies.     

Population size estimation of the pond smelt Hypomesus nipponensis in Lake Kasumigaura and Lake Kitaura,Japan

We estimated the population size of the pond smelt Hypomesus nipponensis at the beginning of the fishing season in Lake Kasumigaura and Lake Kitaura, Japan using two DeLury methods and cohort analysis. A growth curve was estimated on the basis of monthly standard length. The relationship between standard length and weight was utilized for calculating the mean weight on the survey day in a particular month. Total monthly catches in Lake Kasumigaura and Lake Kitaura were calculated using partial monthly pond smelt catch obtained from processing plants located near the lakes between July and December. The total monthly catch was calculated from the total monthly catch weight and the monthly mean weight. The number of boats operating each month, compiled by the Kasumigaura-Kitaura Fisheries Office of Ibaraki Prefecture, was also employed. The estimated initial population sizes were compared with the population level index (PLI) estimated from survey data before the start of the fishing season. No significant differences were detected among the initial population sizes estimated by DeLury method, cohort analysis and PLI. The estimates ranged from 7.4 million to 410 million in Lake Kasumigaura, and from 7.7 million to 44 million in Lake Kitaura.     

Comparison of soil moisture effects on surface conductance between late dry and rainy seasons in an evergreen forest of central Cambodia

Some forests in the Asian monsoon region are reported to transpire actively, even in the late dry season. The evergreen forest in central Cambodia is included in them. The surface conductance was estimated for an evergreen forest in central Cambodia in the late dry (May) and rainy (August) seasons, and the effects of environmental conditions on surface conductance were compared between seasons. The effects of soil moisture did not differ between seasons, indicating that soil water drought in the late dry season does not limit transpiration from the entire forest community. Evergreen forests in central Cambodia are thought to transpire actively in the late dry season.     

Preliminary study of flow regimes and stream water residence times in multi-scale forested watersheds of central Cambodia

To elucidate the water cycle in lowland forests of the Mekong River basin, our research group established four experimental watersheds in the Stung Chinit River basin in Kampong Thom Province, Cambodia. The drainage areas of these experimental watersheds ranged from small (4 km²) to mesoscale (3,659 km²). Here, we present the first preliminary results of our rainfall-discharge observations and analyses of temporal variations of stable isotope ratios in rainfall, stream water, and groundwater. This paper focuses on the following three main topics: annual rainfall, discharge, and water balance; stormflow generation and dominant flow pathways; and flow regimes and stream water residence times. All stream water residence times (τ = 1.7–7.5 months) for the four experimental watersheds were shorter than the residence time of the groundwater (τ = 9.4 months) through the soil and regolith layers, implying that the stream waters consisted of not only the groundwater-flow component, but also younger-aged flow components such as saturation-excess overland flow. The smallest (4 km²; O Toek Loork) watershed had longer residence time (τ = 7.5 months) than the three larger watersheds (126–3,659 km²; τ = 1.7–3.9 months). This may suggest differing contributions of the groundwater and younger-aged flow components in the stream water in each watershed. Our approach of multi-scale watershed observation might better contribute to the needs of physically based models and aid in predictions for ungauged basins.     

Modeling of Stomatal Conductance for Estimating Ozone Uptake of Fagus crenata Under Experimentally Enhanced Free-air Ozone Exposure

We examined a performance of the multiplicative stomatal conductance model to estimate the stomatal ozone uptake for Fagus crenata. Parameterization of the model was carried out by in-situ measurements in a free-air ozone exposure experiment. The model performed fairly well under ambient conditions, with low ozone concentration. However, the model overestimated stomatal conductance under enhanced ozone condition due to ozone-induced stomatal closure. A revised model that included a parameter representing ozone-induced stomatal closure showed better estimation of ozone uptake. Neglecting ozone-induced stomatal closure induced a 20?% overestimation of the stomatal uptake of ozone. The ozone-induced stomatal closure was closely related to stomatal ozone uptake rather than accumulated concentrations of ozone exceeding 40?nmol mol^?1. Our results suggest that ozone-induced stomatal closure should be implemented to stomatal conductance model for estimating ozone uptake for F. crenata. The implementation will contribute to adequate risk assessments of ozone impacts on F. crenata forests in Japan.     

Oxazolone-induced gastrointestinal disorders enhance the oral transmission of AA amyloidosis in mice

Amyloid A (AA) amyloidosis is a lethal disease characterized by systemic AA amyloid deposition, and is reported in many animal species. Despite experiments have shown that AA amyloidosis can be transmitted orally, horizontal transmission and cross-species transmission are concerns, the transmission mechanism has been unknown. In this study, we examined the oral transmission efficiency of AA amyloidosis using oxazolone-induced gastrointestinal disorder mice. As a result, the upper or lower gastrointestinal disorder groups developed more severe amyloid deposition in systemic tissues than the group without gastrointestinal disorders. The results of this study suggest that gastrointestinal damage promotes the oral transmission of AA amyloidosis.     

Evaluation of antixenosis in soybean against Spodoptera litura by dual-choice assay aided by a statistical analysis model: Discovery of a novel antixenosis in Peking

The method for evaluating soybean (Glycine max) antixenosis against the common cutworm (Spodoptera litura) was developed based on a dual-choice assay aided by a statistical analysis model. This model was constructed from the results of a dual-choice assay in which Enrei, a soybean cultivar susceptible to S. litura, was used as both a standard and a test leaf disc for 2nd–5th instar larvae. The statistical criterion created by this model enabled the evaluation of the presence of antixenosis. This method was applied to four soybean varieties, including Tamahomare (susceptible), Himeshirazu (resistant), IAC100 (resistant), and Peking (unknown), as well as Enrei. Subsequently, the degrees of antixenosis were also compared by F-test, followed by maximum likelihood estimation (MLE). According to the results, the antixenosis of Tamahomare, Himeshirazu, and IAC100 was statistically reevaluated and Peking exhibited a novel antixenosis, which was stronger for 3rd–5th instar larvae than for 2nd instar.     

Effect of Biodegradable Gelatin β-Tri Calcium Phosphate Sponges Containing Mesenchymal Stem Cells and Bone Morphogenetic Protein-2 on Equine Bone Defect

This study evaluated the osteoanagenetic effects of administering biodegradable gelatin β-tri calcium phosphate sponges containing mesenchymal stem cells (MSCs) and bone morphogenetic protein-2 (BMP-2) on equine bone defect. Six healthy Thoroughbred horses were used in this study. Horses were anesthetized, and skin incisions were made on all the limbs. Splint bones were exposed and a 1-cm bone defect was created in each exposed bone. Gelatin β-tri calcium phosphate sponges containing MSC and BMP-2 (MSC + BMP-2 sponge), MSC only (MSC sponge), BMP-2 only (BMP-2 sponge), or saline (saline sponge) were implanted into each bone defect at random. Defects were monitored for 16 weeks by radiography followed by computed tomography (CT) and histologic analyses. At 16 weeks, radiographic scores of MSC + BMP-2 sponge-treated defects were significantly higher than those of saline-treated defects (P = .027). Moreover, the CT value of the MSC + BMP-2 sponge group was significantly higher than that of the other groups (P = .027; P = .046; and P = .027, respectively), and the histologic score of the MSC + BMP-2 sponge group was significantly greater than that of the saline sponge group (P = .041). We conclude that MSC + BMP-2 sponge administration to bone defects accelerates bone regeneration in equines.