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.     

Sex,age, or genetic differences related to play behaviors in Japanese Black calves

Play behavior in young animals has been used to evaluate the condition (health) of livestock. We investigated age, sex, and genetic differences of Japanese Black calves in relation to frequency of play behaviors (galloping, leaping, turning, bucking, head butting objects, and head shaking) and examined how these relationships might affect growth during the suckling stage. Locomotor play behaviors (galloping, leaping, turning, and bucking) and head butting objects gradually declined with the age for both sexes. The frequency of head butting was significantly higher in males than females. We found that significant interaction effects (age × MAOA polymorphism) in play behaviors (except head shaking) and the frequencies of locomotor play in calves without the wild‐type allele were significantly higher than those in younger calves (2 and 6 weeks of age). Weight gain was significantly correlated with the frequency of locomotor play in females, but not in males. This study suggests that play in Japanese Black calves gradually declines as they mature and that play may be controlled by variations in the MAOA gene. In addition, the frequency of locomotor play may be an indicator of health in female calves.     

Storage of plug seedlings of tomato under limited fertilisation,and growth,flowering and yield after planting

We stored plug seedlings of tomato (Solanum lycopersicum L. cv. Momotaro) under limited fertilisation (LF) for 12 weeks, and evaluated growth and development during storage and after planting. Seedlings in the LF treatment were grown in substrate containing starter fertiliser and irrigated with water. Controls (CT) were irrigated with nutrient solution. Stem growth, leaf growth, and biomass accumulation slowed or ceased during storage of LF seedlings, while CT seedlings showed increases in these parameters. At 2 weeks after planting, the stem length of LF seedlings was shorter than that of CT seedlings, but the two seedling types had similar numbers of leaves. At harvest, LF and CT plants had similar numbers of leaves under the fruit trusses, but the trusses formed at lower heights in LF than in CT seedlings. LF and CT plants produced similar numbers of fruits and similar yields from the first to third trusses. These results indicate that tomato plug seedlings can be stored for more than 2 months under LF.     

Efficiency of a pedometer device for detecting estrus in standing heat and silent heat in Japanese Black cattle

The usefulness of a radiotelemetric pedometer for estrus detection in standing (ST) heat, or in silent heat without ST events, but in which ovulation is observed, in Japanese Black cattle was investigated. The duration of an increase in steps in ST heat was 11.8 ± 1.3 hr, and it was similar to that of ST events (duration: 10.1 ± 0.8 hr). Even in silent heat, the change pattern and the duration (11.6 ± 0.2 hr) of the period with an increase in steps during estrus were not different compared with ST heat. When artificial insemination (AI) was performed at 15.5 ± 0.6 hr from the onset of estrus detected by the pedometer in ST heat cases, the conception rate was 57.1% (8/14). Furthermore, fertility in cattle that underwent silent heat was evaluated. When AI was performed at 14.4 ± 2.0 hr from the onset of estrus detected by the pedometer, the conception rate was 60% (3/5) in silent heat cases. The overall results suggest that the radiotelemetric pedometer is a valid device for detecting estrus and it can even detect silent heat in Japanese Black cattle. Moreover, even silent heat cattle are fertile when AI is performed at the appropriate time.     

Polymorphism analysis of Fagaceae and DNA-based identification ofFagus species grown in Japan based on therbcL gene

Fagaceae species in Japan were identified by restriction fragment length polymorphism (RFLP) and sequence comparison of a region ofrbcL. Of nine restriction endonucleases used for digestion, three (MspI,RsaI,HaeIII) produced different restriction patterns in Fagaceae. Digestion byMspI yielded four patterns: Fagus species,Castanea crenata, Pasania glabra, and others. Digestion byRsaI andHaeIII afforded two patterns:Fagus species and others. These facts indicate thatCastanea crenata andPasania glabra can be identified byMspI restriction patterns ofrbcL. Sequence comparison of a region of therbcL gene among 20 species of Fagaceae showed that: (1) they could be divided into seven groups; (2) there is a site mutation betweenFagus crenata andF. japonica. The latter indicates that the wood of both Fagus species are identifiable at the species level, which is not the case using conventional methods. This result indicates the possibility of wood identification based on DNA polymorphism in Fagaceae at the intrageneric level.Part of this paper was presented at the 46th annual meeting of the Japan Wood Research Society, Kumamoto, April 3–5, 1996 and the 47th annual meeting of the Japan Wood Research Society, Kochi, April 3–5, 1997     

Binding activity of substituted benzyl derivatives of chloronicotinyl insecticides to housefly‐head membranes,and its relationship to insecticidal activity against the housefly Musca domestica

Variously substituted benzyl derivatives of chloronicotinyl insecticides were synthesized with a wide range of substituents including halogens, NO₂, CN, CF₃ and small alkyl and alkoxy groups at the ortho, meta and para positions, as well as multiple‐substituted benzyl analogues. Their binding activity to the α‐bungarotoxin binding site in housefly (Musca domestica) head membrane preparations was measured. Among the compounds tested, the activity of the meta‐CN derivative was the highest, being 20–100 times higher than those of imidacloprid, acetamiprid and nitenpyram. The synergized insecticidal activity against houseflies was also measured for selected compounds with the metabolic inhibitor, NIA16388 (propargyl propyl phenylphosphonate). For the nitromethylene analogues, including both benzyl and pyridylmethyl analogues, higher binding activity usually resulted in higher insecticidal activity. © 2000 Society of Chemical Industry     

Prediction of the binding mode of imidacloprid and related compounds to house-fly head acetylcholine receptors using three-dimensional QSAR analysis

The binding activity of imidacloprid and related compounds to nicotinic acetylcholine receptors (nAChR) of house flies was measured by use of radioactive α-bungarotoxin as a ligand. Variations in the activity were examined three-dimensionally using comparative molecular field analysis (CoMFA). The CoMFA results suggest that one conformer among the four stable ones is active and provide support for one of the proposed binding models for this class of compound, in which the nitrogen atom of the pyridine ring and the nitrogen atom at the 1-position of the imidazolidine ring interact with the hydrogen-donating and electron-rich sites of nAChR, respectively. The CoMFA field map showed that the nitroimino moiety and a portion of the imidazolidine ring were mainly surrounded by a sterically and electrostatically sensitive region of nAChR. © 1998 Society of Chemical Industry     

Availability of potato protein concentrate as an alternative protein source to fish meal in greater amberjack (Seriola dumerili) diets

Potato protein concentrate (PPC) is a promising candidate as a fish meal (FM) substitute because it has high protein and essential amino acid content. In the present study, we replaced FM in greater amberjack diets with PPC to investigate the effect on growth and feed utilization. Four isonitrogenous, isolipidic and isocaloric experimental diets were prepared by substituting 0, 20, 40 and 60% of FM protein with PPC (Control, P20, P40 and P60 respectively). The in vitro protein digestibility of protein in PPC was 88.8%, relative to 100% protein in the FM. The in vitro protein digestibility of protein in the experimental diets also decreased with increasing PPC and was lowest at 84.2% in P60. After the 7‐week feeding trial, final body weight, weight gain and thermal growth coefficient tended to decrease with increasing PPC and were significantly lower in P60 than in the control (p < .05). Further, fish fed with diets P40 and P60 showed significantly lower feed conversion and protein efficiency ratios than the control group (p < .05). In conclusion, the results suggest that PPC can replace up to 20% of FM in the diet of greater amberjack without compromising the growth performance or feed efficiency.     

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.