Growth and morphologic response of rumen methanogenic archaea and bacteria to cashew nut shell liquid and its alkylphenol components

The growth and morphology of rumen methanogenic archaea (15 strains of 10 species in 5 genera, including 7 strains newly isolated in the present study) and bacteria (14 species in 12 genera) were investigated using unsupplemented in vitro pure cultures and cultures supplemented with cashew nut shell liquid (CNSL) and its phenolic compound components, anti-methanogenic agents for ruminant animals. Growth of most of the methanogens tested was inhibited by CNSL and alkylphenols at different concentrations ranging from 1.56 to 12.5 μg/ml. Of the alkylphenols tested, anacardic acid exhibited the most potent growth inhibition. Three gram-negative bacterial species involved in propionate production were resistant to CNSL and alkylphenols (>50 μg/ml). All the methanogens and bacteria that were sensitive to CNSL and alkylphenols exhibited altered morphology; disruption of the cell surface was notable, possibly due to surfactant activity of the tested materials. Cells division was inhibited in some organisms, with cell elongation and unclear septum formation observed. These results indicate that CNSL and alkylphenols, particularly anacardic acid, inhibit both rumen bacteria and methanogens in a selective manner, which could help mitigate rumen methane generation.     

Effect of activation treatments on actin filament distribution and in vitro development of miniature pig somatic cell nuclear transfer embryos

In the present study, we investigated the effect of activation treatments on the actin filament distribution and in vitro development of somatic cell nuclear transfer (SCNT) embryos in miniature pigs. We combined three activation methods, ionomycin (ION), electrical stimulation (ES), and cycloheximide treatment (CH), to prepare seven activation treatments (ION, ES, CH, ION + CH, ION + ES, ES + CH and ION + ES + CH). First, we investigated the activation rate of oocytes and in vitro development of parthenotes. The activation rates of the oocytes in the ION, ES, CH, ION + CH, ION + ES, ES + CH, and ION + ES + CH groups were 42.9, 51.3, 0.0, 82.1, 80.6, 78.1 and 78.6%, respectively, showing that the rates of the combined treatment groups were significantly higher (P<0.05) than those of the single treatment groups. Although there were no significant differences in the activation rates of the combined treatment groups, the developmental rate to blastocysts in the ION + CH treatment group (36.1%) was significantly higher (P<0.05) than the other combined treatment groups (14.6-24.7%). Subsequently, we investigated the in vitro development and distribution of microfilaments in SCNT embryos. The developmental rate to blastcysts of the SCNT embryos in the ION + CH treatment group (11.3%) was significantly higher (P<0.05) than in the ES and ION + ES + CH treatment groups (4.5 and 5.2%, respectively). The rate of normal actin filament distribution in the SCNT embryos activated with ION + CH was significantly higher (P<0.05) than those activated with ES or ION + ES + CH treatment (63.3 vs. 46.8 or 46.4%). In addition, the fragmentation rate of the SCNT embryos activated with ION + CH was significantly lower (P<0.05) than those activated with ION + ES + CH (14.9 vs. 26.1%). The present results suggest that an activation treatment of ionomycin combined with cycloheximide may avoid physical damage to microfilaments and result in improved subsequent development of miniature pig SCNT embryos.     

Large-scale survey of frequency of forest walking and related factors in a Japanese population inhabiting a large city, and comparison of an urban area and a rural area

The frequency of forest walking among the general population is a major topic of study in forest science. The objectives of this study were threefold: to assess the frequency of forest walking among Japanese residents of Nagoya, a large city; to evaluate differences among frequency of forest walking by participants from a large city, an urban area (Shizuoka; 4,666 participants), and a rural area (Yakumo Town, Hokkaido; 397 participants) in previous studies; and to examine factors related to frequency of forest walking. The survey, by self-administered questionnaire, in the major city of Nagoya was conducted between June 2008 and May 2010. In all, 5,158 participants (M/F, 1,466/3,692; mean age ± SD [range], 52.5 ± 10.3 [35–69] years) were included in the analysis. The proportions of frequency of forest walking ≥ once/month and ≥ once/year were 10.9 % (M/F, 15.1/9.3 %) and 46.1 % (51.0/44.1 %), respectively. In multiple logistic regression analysis, significant differences were noted among study sites for the adjusted odds ratio for frequency of forest walking. Overall, the order of highest to lowest frequency of forest walking was Shizuoka > Nagoya > Yakumo. Factors related to frequency of forest walking were common among the three study sites. Higher frequency of forest walking was associated with male sex and older age; the most relevant factor related to frequency of forest walking was its enjoyment level. Further studies will be required to clarify why these factors are related to frequency of forest walking.     

Influence of Rice Proteins on Eating Quality of Cooked Rice and on Aroma and Flavor of Sake

An improved method for the extraction of storage proteins from rice endosperm under conditions safe for producing food was developed. The contribution of the protein extracts to the eating quality of cooked rice and to the aroma and flavor of sake was examined. Sensory analysis was performed to evaluate the eating quality of cooked rice enriched with the protein extracts. Prolamin‐enrichment increased the hardness of cooked rice, and glutelin‐enrichment degraded the appearance of cooked rice. Physical analyses confirmed that prolamin‐enrichment changed, whereas the glutelin‐enrichment did not change the physical properties of cooked rice. Light and scanning electron microscopy of rice protein extracts revealed small particles of the prolamin extract and larger aggregated particles of the glutelin extract; these features remained after heating. The aroma and flavor of sake were negatively affected by the addition of the protein extracts. Especially, addition of prolamin significantly lowered the evaluation score of the aroma obtained by sensory analysis.     

Molecular characteristics of horse phospholipase C zeta (PLCζ)

A sperm‐specific phospholipase C (PLC), PLCzeta (PLCζ), is thought to underlie the initiation of calcium ([Ca²⁺]_i) oscillations that induce egg activation in mammals. In large domestic species, only bovine, porcine and recently equine PLCζ have been cloned, and the physiological functions of these molecules have not been fully characterized. Here, we evaluated the physiological functions of equine PLCζ (ePLCζ) in mouse oocytes. ePLCζ was cloned from testis using RT‐PCR. The expression of ePLCζ messenger RNA was confirmed in testis but not in other tissues. Microinjection of ePLCζ complementary RNA (cRNA) into mouse oocytes induced long‐lasting [Ca²⁺]_i oscillations, and most of the injected oocytes formed pronuclei (PN). The injection of cRNAs encoding horse, mouse, human and cow PLCζ into mouse oocytes showed that ePLCζ had the highest [Ca²⁺]_i oscillation‐inducing activity among the species tested. Mutation of D202R, which renders the protein inactive, abrogated the activity of ePLCζ. The nuclear translocation ability of ePLCζ was defective when expressed in mouse oocytes. Taken together, our findings show for the first time that ePLCζ has highest activity of the mammalian species studied to date. Our findings will be useful for the improvement of reproductive technologies in the horse.