Heat shock-derived reactive oxygen species induce embryonic mortality in in vitro early stage bovine embryos

Heat shock is known to increase the mortality of early stage embryos, but the exact mechanism is unclear. In the present study, we investigated the possibility that the increased mortality is caused by heat shock-generated reactive oxygen species (ROS). The level of ROS was controlled by using beta-mercaptoethanol (beta-ME), a scavenger of ROS. In vitro-produced 8-cell stage embryos were cultured at 38.5 C or heat-shocked by exposure to 41 C for 6 h with 0, 10 and 50 microM beta-ME. Intracellular ROS levels were measured by a fluorescent dye, 2',7'-dichlorodihydrofluorescein diacetate (DCHFDA), and intracellular reduced form of glutathione (GSH) contents were estimated by another fluorescent dye, 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin. Total glutathione content was estimated by the glutathione recycling assay. On day 8 after insemination, heat shock decreased the percentage of embryos that developed to the blastocyst stage and increased intracellular ROS levels, but there was no significant effect on the GSH and total glutathione contents. In contrast, beta-ME significantly decreased ROS levels in heat-shocked embryos and increased the GSH and total glutathione concentrations. Ten microM beta-ME significantly improved the viability of heat-shocked embryos. beta-ME caused no detrimental effects when it was added at normal culture temperature (38.5 C). These results indicate that ROS is the primary cause of increased embryonic mortality in heat-shocked early stage embryos.     

The effect of intermittent administration of sustained release isosorbide dinitrate (sr-ISDN) in rats with volume overload heart

Recently, it has been reported that intermittent administration of nitrate, with a nitrate-free interval of 10 to 12 hr eliminated expression of tolerance, and maintained its hypotensive effect. In the present study, we evaluated whether nitrate tolerance developed or not with an intermittent administration of sr-ISDN (5 mg/kg/ once a day) in Wistar rats. The effect of this administration protocol for sr-ISDN on the volume overload heart model, aortovenous fistula, was also examined. Furthermore, blood pressure was monitored by radio telemetry during sr-ISDN (5 mg/kg/once a day) administration. Nitrate tolerance did not develop, and eccentric hypertrophy due to volume overload was moderated by sr-ISDN administration. Sr-ISDN administration maintained blood pressure lower level than the placebo group. In conclusion, prolonged intermittent administration of sr-ISDN maintained its hypotensive effect during the entire experiment period, without developing tolerance, and moderated efferent hypertrophy with attenuated volume overload.     

DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle

Many observations have been made on cloned embryos and on adult clones by somatic cell nuclear transfer (SCNT), but it is still unclear whether the progeny of cloned animals is presenting normal epigenetic status. Here, in order to accumulate the information for evaluating the normality of cloned cattle, we analyzed the DNA methylation status on satellite I region in blastocysts obtained from cloned cattle. Embryos were produced by artificial insemination (AI) to non‐cloned or cloned dams using semen from non‐cloned or cloned sires. After 7 days of AI, embryos at blastocyst stage were collected by uterine flushing. The DNA methylation levels in embryos obtained by using semen and/or oocytes from cloned cattle were similar to those in in vivo embryos from non‐cloned cattle. In contrast, the DNA methylation levels in SCNT embryos were significantly higher (P < 0.01) than those in in vivo embryos from non‐cloned and cloned cattle, approximately similar to those in somatic cells used as donor cells. Thus, this study provides useful information that epigenetic status may be normal in the progeny of cloned cattle, suggesting the normality of germline cells in cloned cattle.     

Contribution of Adventitious Roots to Water Use Strategy of Juniperus sabina in a Semiarid Area of China

duniperus sabina Linnaeus, an evergreen shrub with prostrate life form, can effectively prevent sand moving and is an important tree species for reforestation in semiarid areas of China. It has laterally distributed adventitious roots and a deeply distributed main root system. To detect water movement between the main root system and adventitious roots, we adopted heat pulse sensors using the Heat Ratio Method, a high precision method for measuring low sap flow rates. Two sensors were implanted in each individual in the stem between the main root system and adventitious roots, and another two in lateral stems distal to all the roots. Positive sap flows during nighttime, even under saturated air moisture conditions, were detected only between the main root system and adventitious roots under drought conditions, and the rate of flow increased as drought progressed and decreased or disappeared after rain events. The results demonstrated the existence of water movement from the main root system to adventitious roots, and combined with the high contribution of nighttime sap flow to transpiration （11%-16%） the results indicate that it also involves the process of hydraulic lift, a water movement from moist subsoil to dry surface soils. Integrated water use strategy between the main root system and adventitious roots via the process of hydraulic lift of soil water maximizes water acquisition efficiency from both subsoil water and water from rain pulses on the soil surface; this increases survivability in the water-limited environment of semiarid areas.     

Establishment of a Method to Measure Length of the Ulnar Nerve and Standardize F-wave Values in Clinically Normal Beagles

We designed a new method of measuring the length of the ulnar nerve and determining standard values for F-wave parameters of the ulnar nerve in clinically normal beagles. Nerve length must be precisely measured to determine F-wave latency and conduction velocity. The length of the forelimb has served as the length of the ulnar nerve for F-wave assessments, but report indicates that F-wave latency is proportional to the length of the pathway traveled by nerve impulses. Therefore, we measured the surface distance from a stimulus point to the spinous process of the first thoracic vertebra (nerve length 1) and the anterior horn of the scapula (nerve length 2) as landmarks through the olecranon and the shoulder blade acromion. The correlation coefficients between the shortest F-wave latency and the length of nerves 1, 2 or the forelimb were 0.61, 0.7 and 0.58. Nerve length 2 generated the highest value. Furthermore, the anterior horn of the scapula was easily palpated in any dog regardless of well-fed body. We concluded that nerve length 2 was optimal for measuring the length of the ulnar nerve.     

Reports on establishing an ex situ site for ‘beautiful’ vavilovia (Vavilovia formosa) in Armenia

Vavilovia (Vavilovia Fed.) is one of the five genera in tribe Fabeae and consists of only one species, ‘beautiful’ vavilovia (Vavilovia formosa (Stev.) Fed.). The main centre of distribution is the Central and Eastern Caucasus, with a disjunct distribution among high alpine areas in the region, extending as far as West Turkey, Lebanon and Iran. In Armenia, in situ studies on Vavilovia started in the late 1930s. In July and August 2009, three expeditions were conducted to two locations: two to the Ughtasar Mountain and one to the Geghama Mountains. The first expedition to Ughtasar resulted in fresh plant collections and soil analysis for one of the sites. The expedition to Geghama established the existence of Vavilovia in the region of Lake Aknalitch. The second expedition to Ughtasar provided immature fruits and seeds. Collected plant material was transplanted into the Flora and Vegetation of Armenia plot of the Yerevan Botanic Garden established in 1940. Today, along with other plants the plot contains more than 200 species of wild relatives of cultural plants from 130 genera, including indiginous species of tribe Fabeae such as Vavilovia. The transplanted plants will continue to be monitored to see if the plants go on to successfully flower and set seed or whether further sites, possibly at higher altitudes might need to be tested to meet the long term conservation requirements of this iconic legume. These co-ordinated efforts provide a good example of an ex situ conservation strategy for Vavilovia formosa, which, if successful will improve access and utility for the whole legume research community.     

Megaesophagus was complicated with Billroth I gastroduodenostomy in a cat

A seven-year-old, female, domestic short hair cat was presented with a history of chronic anorexia. Radiographic examination revealed a large space-occupying calcified mass in the abdominal cavity. The mass was located in pylorus and did not extend into the duodenum and surrounding tissues. Billroth I gastroduodenostomy was conducted to remove the mass. Histopathological examination of the mass showed a lymphoma. Although Recovery following the operation was excellent, the patient showed intermittent vomiting unrelated to feeding. Radiographical examination revealed a megaesophagus, which was assumed to be a complication of the Billroth I procedure, since the condition was not observed before the procedure.