Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Effects of AI protocol and time interval from onset of estrus to AI on conception rate in Japanese Black cows

Two different artificial insemination (AI) protocols were investigated to determine the effect of the time interval from the onset of estrus (as detected by a pedometer) to the AI on the conception rate in Japanese Black cows. Seventy‐three cows were divided into two groups that received AI either after the induction of synchronized ovulation (synchronized AI group; n = 26) or at the time of observed estrus (control AI group; n = 47). Each group was further divided into two subgroups, which were artificially inseminated either 0–12 h (early AI group; n = 21) or 12–24 h (late AI group; n = 52) after the onset of estrus. There was no significant difference in the conception rate between the synchronized AI and control AI groups. The AI protocol and the detection of estrus using a threshold of counted steps (as measured by a pedometer) in this study could be effective for planned reproduction management without the observation of standing estrus in Japanese Black cows.     

Influence of Equine Conformation on Rider Oscillation and Evaluation of Horses for Therapeutic Riding

To obtain basic knowledge about selecting horses for therapeutic riding, the influence of equine conformation on rider oscillation and relationships between these factors and the evaluation on horses as the therapeutic riding were studied. Thirty-five riding horses were used. Equine conformation was estimated by 24 indices. Rider oscillation was measured by an accelerometer fixed at the rider’s waist. The spatial position of the oscillation was estimated by a double integration of the acceleration. Horses were evaluated for therapeutic riding by a Riding for the Disabled Association instructor as a rider. Evaluations were on a scale of 1 to 5, with 5 being the highest score for 27 items. Horses were classified into 4 groups: the short and narrow (SN), short and wide (SW), tall and narrow (TN), and tall and wide (TW). The frequencies of rider oscillation both at walk and trot were higher (P<0.01), and the vertical (P<0.01) and longitudinal (P<0.05) amplitudes at trot were smaller, on short horses than on tall horses. The vertical amplitude at walk was smaller (P<0.05) and the lateral amplitude at trot was larger (P<0.01) on wide horses than on narrow horses. Short horses could be used for the rider who requires side walkers. Wide horses could be used for relieving muscular tension and for the rider who could not maintain good balance on the horse. Short and wide horses should be suitable for therapeutic riding.     

Mean retention time of digesta in the different segments of the equine hindgut

Six Thoroughbred horses were used to determine the mean retention time (MRT) of digesta in the different segments of the hindgut. The horses were fed timothy hay or silage in equal amounts (1.6% bodyweight [BW]/day) every 3 h/day. Hay or silage labeled with the rare earth elements cerium, dysprosium, neodymium, ytterbium, lanthanum, samarium and praseodymium, were fed to the horses 36, 30, 24, 18, 12, 6 and 3 h before slaughter, respectively. Just after slaughter, digesta samples in the different segments were collected and determined for the contents of the rare earth elements. Using these data, a method to calculate the MRT in the different segments was proposed. There were no significant differences between diets in the MRT of each segment. The averaged MRT of hay and silage in the cecum, right ventral colon, left ventral colon, left dorsal colon, right dorsal colon and small colon was 2.9, 3.1, 5.9, 1.0, 4.0, and 4.0 h, respectively. The dry matter weight of the digesta was related to the MRT in the right ventral colon (r = 0.94, P = 0.005), left ventral colon (r = 0.87, P = 0.03), left dorsal colon (r = 0.98, P < 0.001) and right dorsal colon (r = 0.95, P = 0.004), but it was not related to the MRT in the cecum and small colon.     

Identification of quantitative trait loci for powdery mildew resistance in highly resistant cucumber (Cucumis sativus L.) using ddRAD-seq analysis

Powdery mildew, caused by Podosphaera xanthii (syn. Sphaerotheca fuliginea ex Fr. Poll.), is one of the most economically important foliar diseases in cucumber (Cucumis sativus L.). Cucumber parental line ‘Kyuri Chukanbohon Nou 5 Go’, developed from weedy cucumber line CS-PMR1, is highly resistant to powdery mildew and is promising breeding material. We performed quantitative trait locus (QTL) analysis using double-digest restriction-site-associated DNA sequencing (ddRAD-Seq) in a population from a cross between ‘Kyuri Chukanbohon Nou 5 Go’ and the Japanese native cultivar ‘Kaga-aonaga-fushinari’, which is susceptible to powdery mildew. The resistance of the population and its parents was evaluated using leaf disc assays and image analysis. We detected one major QTL on Chr. 5 that was effective at both 20°C and 25°C and one minor QTL on Chr. 1 effective at 20°C. We detected two additional QTLs in subpopulation: one on Chr. 3 effective at 20°C and one on Chr. 5 effective at both 20°C and 25°C in a position different from the major QTL. The resistance alleles at all four QTLs were contributed by ‘Kyuri Chukanbohon Nou 5 Go’. The results of this study can be used to develop practical DNA markers tightly linked to genes for powdery mildew resistance.     

Feeding activity rhythm in Japanese sea catfish <Emphasis Type="Italic">Plotosus japonicus</Emphasis>

To examine the feeding activity characteristics of Japanese sea catfish Plotosus japonicus, access to feed box (feeding activity) and the number of food pellets consumed (food intake) were recorded under laboratory conditions. When fed ad libitum under a 12:12 h light–dark (LD) cycle, all fish exhibited feeding activity and consumed food during the dark period. Feeding activity increased in the presence of food, and this increased activity level continued for several days after food removal. During restricted food availability within the light period of the LD cycle, seven of nine fish exhibited food-anticipatory activity (FAA) both before and after the 12:00 food-restricted time. This FAA persisted under constant light conditions (in nine of nine fish). The average circadian free-running period was 24 h. These results suggest that P. japonicus exhibits a nocturnal feeding activity rhythm, which may be synchronized by a food-restricted time.     

Effect of arsenic on concentrations and translocations of mineral elements in the xylem of rice

By using Particle-Induced X-ray Emission (PIXE) technique, the effect of arsenic (As) on the mineral contents and translocation in the xylem of rice (Oryza sativa L. cv. ‘Akihikari’) was studied. The results suggest that exogenous As increased the concentrations of phosphorus (P), calcium (Ca) magnesium (Mg), sulfur (S), and manganese (Mn) in xylem, while the concentrations of potassium (K) remained unchanged. The highest concentration of As to the rice roots did not have any clear effect on the translocation of P, Ca, S, and chlorine (Cl) in the xylem, indicating that the increasing concentrations of the minerals may be due to a condensation effect, resulting from the repression of water movement in xylem by As-toxicity. Among the metal micronutrients, As decreased the concentrations and translocations of iron (Fe), zinc (Zn), and copper (Cu).     

Effects of tightening speed on torque coefficient in lag screw timber joints with steel side plates

To investigate the effects of tightening speed on the torque coefficient in lag screw timber joints with steel side plates, tightening tests were conducted on main timber members made from Cryptomeria japonica, Chamaecyparis obtusa and Pseudotsuga menziesii, under four tightening speed conditions (1, 4, 10, and 20 rpm). Major stick-slip behavior was observed in C. obtusa based on the relationship of tightening angle with clamp force, tightening torque, and thread torque at tightening speeds of 1 and 4 rpm. In addition, tightening speed’s effects on the torque coefficient (K) varied depending on the wood species of the main member. In P. menziesii, K was not affected by the tightening speed: the ratio of torque expended on tightening was 25% on average, and the ratio of torque expended on bearing surface friction was higher than the ratio of torque expended on thread friction.     

Effect of timing and type of supplementary grain on herbage intake,nitrogen utilization and milk production in dairy cows grazed on perennial ryegrass pasture from evening to morning

The present study aimed to clarify the effect of timing and type of supplementary grain in grazing dairy cows on herbage dry matter intake (HDMI), nitrogen utilization and milk production. Eight lactating cows were allowed to graze from evening to morning during three seasonal periods (spring, summer, autumn). They were randomly allocated to four treatments (timing: pre‐ (Pre) or post‐grazing (Post), for large grain allotments consisting of 75% of daily grain offered; grain type: barley or corn) in 4 × 4 Latin square designs in each period. In the spring period, HDMI was greater for cows fed corn than those fed barley (P = 0.005), whereas cows in the Pre treatment had a similar HDMI, higher (P = 0.049) urinary purine derivative concentration and greater (P = 0.004) milk yield compared with cows in the Post treatment. In the summer and autumn periods, timing treatments did not affect HDMI, nitrogen utilization or milk production, but cows supplemented with barley had higher urinary purine derivatives concentration (P < 0.05) and milk yield (P < 0.05) compared with those supplemented with corn. The results indicate that large grain allotments immediately before evening grazing during early grazing seasons increased ruminal microbial protein synthesis and milk production without reducing HDMI regardless of grain type.