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.     

Molecular detection of Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum' Infection in cats by direct PCR using whole blood without DNA extraction

Detection of hemotropic Mycoplasma spp. infection was attempted in cats by PCR using whole blood without DNA extraction. A total 46 of 54 (85%) cats with suspected Mycoplasma spp. infection showed a positive reaction, corresponding completely with the results of standard PCR testing. The direct PCR assay was sensitive enough to detect more than 0.0061% parasitemia for ;C. M. haemominutum' and 0.0075% parasitemia for M. haemofelis. These data indicate that the direct PCR assay might be sufficient for use as a tool in clinical examinations.     

Hypereosinophilic syndrome in two cats

Two cats showing chronic vomiting, diarrhea and weight loss were found to have leukocytosis with marked eosinophilia. Both cats were diagnosed with hypereosinophilic syndrome by the findings of increased eosinophils and their precursors in the bone marrow, eosinophilic infiltration into multiple organs, and exclusion of other causes for eosinophilia. Although cytoreductive chemotherapy with hydroxycarbamide and prednisolone was performed, these two cats died 48 days and 91 days after the initial presentation.     

Effects of follicle‐stimulating hormone followed by gonadotropin‐releasing hormone on embryo production by ovum pick‐up and in vitro fertilization in the river buffalo (Bubalus bubalis)

In this study, we examined the effects of superstimulation using follicle‐stimulating hormone (FSH) followed by gonadotropin‐releasing hormone (GnRH) on buffalo embryo production by ultrasound‐guided ovum pick‐up (OPU) and in vitro fertilization (IVF). Nine Murrah buffaloes were subjected to OPU‐IVF without superstimulation (control). The morphologies of the oocytes collected were evaluated, and oocytes were then submitted to in vitro maturation (IVM). Two days after OPU, same nine buffaloes were treated with twice‐daily injections of FSH for 3 days for superstimulation followed by a GnRH injection. Oocytes were collected by OPU 23–24 hr after the GnRH injection and submitted to IVM (the superstimulated group). The total number of follicles, number of follicles with a diameter > 8 mm, and number of oocytes surrounded by multi‐layered cumulus cells were higher in the superstimulated group than in the control group (p ≤ 0.05). After IVF, the percentages of cleavage and development to blastocysts were higher in the superstimulated group than in the control group (p < 0.05). In conclusion, superstimulation improved the quality of oocytes and the embryo productivity of OPU‐IVF in river buffaloes.     

Variation in Virulence of Plasmodiophora brassicae in Japan Tested with Clubroot-resistant Cultivars of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

The differential hosts of Williams (1966) and the European Clubroot Differential (ECD) (Buczacki et al., 1975) have been used commonly to identify populations of Plasmodiophora brassicae, which causes clubroot disease in Brassica crops. However, some of these hosts showed intermediate and fluctuating scores to most populations from Japan. Therefore, these hosts could not be used to provide a clear classification in Japan. We have tried to clarify the genetic diversity in pathogenicity of P. brassicae in Japan using Japanese clubroot-resistant (CR) F₁ hybrid (F₁) cultivars and lines of Brassica rapa. The responses of some CR F₁ cultivars were very clear. Four groups of field populations in Japan were recognized using the CR F₁ cultivars. The clear response obtained here may depend largely on the genetic purity of the F₁ cultivars. Moreover, it is possible to classify some of these Japanese populations into the same race using the Williams set and ECD 01 to ECD 05. The present differential hosts may be useful in the study of European populations of P. brassicae. The response of the differential hosts suggests that there are several major CR genes in B. rapa. It is suggested that pyramiding CR genes would be useful in breeding CR cultivars that can overcome the breakdown of the present CR cultivars of Chinese cabbage.     

Effect of water temperature on the feeding activity and the resultant mercury levels in the muscle of cultured bluefin tuna Thunnus orientalis (Temminck and Schlegel)

Tuna muscle often contains high levels of mercury, and fish samples with mercury concentrations ten times higher than the specified safety standards have been reported. Here, we report on the relationship between water temperature and the concentration of mercury in the tail muscle tissue of cultured bluefin tuna Thunnus orientalis. The fish used in this study were cultured at Fisheries Laboratory of Kinki University (Amami Experimental Station, Kagoshima, Japan). One hundred fish weighing 26.2–89.4 kg were selected for analysis between February 2007 and January 2008. Water temperature during rearing ranged from 21 to 29 °C. The total mercury levels were measured using the reduction vaporizing atomic absorption method after acid digestion. Body weight increased approximately 1.5 times that observed in a previous study, despite feeding activity either being the same or less than that observed previously. The average mercury concentration in white muscle was 0.353 mg kg^?1, remaining almost constant and independent of body growth. Unlike previous studies, seasonality was not observed in this study. Based on these findings, water temperatures within a certain range were considered to stabilize feeding activity and increase feeding efficiency. Consequently, water temperature is considered to have a moderating effect on seasonal fluctuations in muscle mercury concentrations in cultured bluefin tuna.     

Effects of a probiotic bacterial <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> dietary supplement on the crowding stress response of juvenile Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis>

To test the effects of probiotic bacteria against crowding stress, juvenile Nile tilapia Oreochromis niloticus were fed commercial feed supplemented with 1 × 10¹⁰ cfu/g pellets of Lactobacillus rhamnosus for 3 weeks. The fish were confined and subjected to crowding for 7 and 14 days after which their physiological condition was investigated. The administration of probiotic prophylaxis improved growth performance even in the stressed fish. After 7 days of stress exposure, fish receiving probiotic-supplemented feed showed proactive behavior and coping responses to the stressor, as evidenced by elevated plasma glucose levels and osmolality, stabilized plasma electrolytes, and a higher RNA:DNA ratio. Fish stressed for 7 days and fed normal commercial diet showed impairment of plasma electrolytes; after 14 days, the plasma osmolality, electrolytes, glucose, and RNA:DNA ratio decreased, indicating physiological maladaptation to the stressor. The growth rate was also found to be reduced, suggesting that energy demand exceeded the energy available from metabolism and dietary uptake. This latter effect was not observed in fish fed the probiotic-supplemented diet. The results suggest that probiotic bacteria prophylaxis may increase energy availability for metabolic support of the crowding stress response and improve the stress coping capacity of fish.     

How to determine the appropriate mortality in experimental larval rearing?

Survival in larval rearing experiments is difficult to estimate due to accidental losses and periodic sampling. The number of sampled fish can be a large proportion of the stocked ones, making it difficult to calculate the overall survival rate and mortality coefficient as this is based on the initial number. Here, a new method of calculating survival is proposed using the mortality coefficient. When the initial stocking density and sampled and final numbers are known, and assuming that mortality coefficient is constant, the final number of fishes can be represented by the formula N _t = e^−mt (N ₀ − ΣN _Sne ^mdn), where t is rearing period (days), N ₀ indicates initial number, N _t indicates the survival number at t days of rearing, m is the natural mortality coefficient, N _Sn is the sampled number in the nth sampling, and dn is the rearing period until removal of the nth sample. The provisional mortality coefficient is calculated from initial and final stocking numbers. Then values for the natural mortality coefficient are substituted into the formula with successive approximation. The coefficient, which most closely approximates the actual survival, is determined as the best fit natural mortality coefficient. Examples of larval experiments are provided to demonstrate the method and show that survival is often underestimated using traditional methods.     

Inhibition of transient receptor potential vanilloid type 1 through α2 adrenergic receptors at peripheral nerve terminals relieves pain

The activation of α₂ adrenergic receptors contributes to analgesia not only in the central nervous system but also in the peripheral nervous system. We reported that noradrenaline inhibits the activity of transient receptor potential vanilloid 1 (TRPV1) evoked by capsaicin through α₂ receptors in cultured rat dorsal root ganglion (DRG) neurons. However, it is unclear whether activation of TRPV1 expressed in peripheral nerve terminals is inhibited by α₂ receptors and whether this phenomenon contributes to analgesia. Therefore, we examined effects of clonidine, an α₂ receptor agonist, on several types of nociceptive behaviors, which may be caused by TRPV1 activity, and subtypes of α₂ receptors expressed with TRPV1 in primary sensory neurons in rats. Capsaicin injected into hind paws evoked nociceptive behaviors and clonidine preinjected into the same site inhibited capsaicin-evoked responses. This inhibition was not observed when clonidine was injected into the contralateral hind paws. Preinjection of clonidine into the plantar surface of ipsilateral, but not contralateral, hind paws reduced the sensitivity to heat stimuli. Clonidine partially reduced formalin-evoked responses when it was preinjected into ipsilateral hind paws. The expression level of α_2C receptor mRNA quantified by real-time PCR was highest followed by those of α_2A and α_2B receptors in DRGs. α_2A and α_2C receptor-like immunoreactivities were detected with TRPV1-like immunoreactivities in the same neurons. These results suggest that TRPV1 and α₂ receptors are coexpressed in peripheral nerve terminals and that the functional association between these two molecules causes analgesia.