Preincubation with green tea polyphenol extract is beneficial for attenuating sperm injury caused by freezing‐thawing in swine

Polyphenols (PFs) extracted from green tea, known to be potent anti‐oxidants, have been reported to be effective in increasing the motility and viability of mammalian sperm, preserved in a liquid form. Therefore, we tested whether PFs might also be effective for maintaining the integrity of frozen‐thawed boar spermatozoa. Ejaculates, collected from Clawn miniature pigs, were diluted in a semen extender containing various amounts of PFs (0, 0.01, 0.05, 0.1 and 0.2% w/v) and then stored at 15°C overnight. The semen samples were processed, using the straw freezing procedure, and then frozen in liquid nitrogen. After rapid thawing at 40°C, the spermatozoa were subjected to several assays to evaluate semen quality. Spermatozoa frozen in a medium containing 0.01% w/v PFs exhibited significantly (P < 0.05) higher degrees of post‐thawed viability and acrosomal integrity than those stored in the absence of PFs. However, no change in the mitochondrial activity was noted between the two groups. The inclusion of 0.01% PFs in the semen extender was significantly (P < 0.05) effective in increasing both the rates of monospermic oocyte formation and of blastocyst formation. These findings indicate that preincubation with the semen extender, containing 0.01% PFs prior to freezing, exerts a protective effect on boar sperm by preventing injuries associated with freezing‐thawing.     

A new rolling culture‐based in vitro fertilization system capable of reducing polyspermy in porcine oocytes

The high incidence of polyspermy is one of the major obstacles during in vitro fertilization (IVF) in pigs. To overcome this, we developed a novel IVF method, which involves constant rotation. Oocytes matured in vitro were mixed with spermatozoa (0.2 × 10⁵ sperm/mL) in an IVF medium (200 μL) using a 200 μL PCR tube. This tube was then rotated at 1 rpm for 6 h at 38.5°C in a rotation mixer (experimental group). A second PCR tube was simultaneously cultured without rotation (control group). The rate of polyspermy was evaluated 12 h after insemination and was significantly (P < 0.05; 21.0% vs. 48.3%) lower in the experimental group than in the control group. Sperm penetration rate was similar in oocytes from the experimental and control groups (75.2% vs. 83.1%). However, monospermic fertilization rate of the oocytes was significantly (P < 0.05; 44.8% vs. 21.2%) higher in the experimental group than in the control group. Furthermore, the rate of blastocyst formation (30.1% vs. 20.8%) increased in the experimental group, as compared to the control group. This present system will contribute to increase the efficacy of blastocyst production through reduction of polyspermic penetration.     

Spatial kernel models capturing field heterogeneity for accurate estimation of genetic potential

According to Fisher’s principles, an experimental field is typically divided into multiple blocks for local control. Although homogeneity is supposed within a block, this assumption may not be practical for large blocks, such as those including hundreds of plots. In line evaluation trials, which are essential in plant breeding, field heterogeneity must be carefully treated, because it can cause bias in the estimation of genetic potential. To more accurately estimate genotypic values in a large field trial, we developed spatial kernel models incorporating genome-wide markers, which consider continuous heterogeneity within a block and over the field. In the simulation study, the spatial kernel models were robust under various conditions. Although heritability, spatial autocorrelation range, replication number, and missing plots directly affected the estimation accuracy of genotypic values, the spatial kernel models always showed superior performance over the classical block model. We also employed these spatial kernel models for quantitative trait locus mapping. Finally, using field experimental data of bioenergy sorghum lines, we validated the performance of the spatial kernel models. The results suggested that a spatial kernel model is effective for evaluating the genetic potential of lines in a heterogeneous field.     

Multiplex PCR method for differentiating highly pathogenic Yersinia enterocolitica and low pathogenic Yersinia enterocolitica,and Yersinia pseudotuberculosis

A multiplex PCR method for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as the highly pathogenic Y. enterocolitica, including serotype O8, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis, was developed. Four primer pairs were chosen to detect the genes fyuA, ail, inv, and virF, responsible for the virulence in pathogenic Yersinia species. Under the multiplex PCR conditions, the unique band patterns for the highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis were generated from Yersinia strains. The detection limit of this method was 10¹–10³ CFU per reaction tube. This multiplex PCR method could detect highly pathogenic Y. enterocolitica O8 from the wild rodent fecal samples that were culture-positive. Therefore, the new multiplex PCR method developed in this study is a useful tool for rapid and sensitive diagnosis, distinguishing three pathogenic Yersinia groups.     

Visualization of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> cells during biocontrol of bacterial wilt disease in tomato with <Emphasis Type="Italic">Pythium oligandrum</Emphasis>

The biocontrol agent Pythium oligandrum (PO) can suppress bacterial wilt caused by Ralstonia solanacearum (RS) in tomato. To understand the primary biocontrol mechanisms of bacterial wilt by PO, we pretreated tomato plants with sterile distilled water or preinoculated them with PO, followed by inoculation with RS, then observed PO and RS in fixed sections of tomato tissues using a confocal laser-scanning microscope and fluorescence labeling until 14 days after the inoculation with RS. Horizontal and vertical movement of RS bacteria was frequently observed in the xylem vessels of roots and stems of tomato plants (cv. Micro-Tom) that had not been inoculated with PO. In plants that were preinoculated with PO, the movement of RS was suppressed, and bacteria appeared to be restricted to the pit of vessels, a reaction similar to that observed in resistant rootstocks. PO colonization was mainly observed at the surfaces of taproots, the junctions between taproots and lateral roots, and the middle sections of the lateral roots. PO was not observed near wound sites or root tips where RS tended to colonize. However, RS colonization was significantly repressed at these sites in PO preinoculated plants. These observations suggest that the induction of plant defense reactions is the main mechanism for the control of tomato bacterial wilt by PO, not direct competition for infection sites.     

Characterization of Antioxidant Activity of Heated Mycosporine-like Amino Acids from Red Alga Dulse Palmaria palmata in Japan

We recently demonstrated the monthly variation and antioxidant activity of mycosporine-like amino acids (MAAs) from red alga dulse in Japan. The antioxidant activity of MAAs in acidic conditions was low compared to that in neutral and alkali conditions, but we found strong antioxidant activity from the heated crude MAA fraction in acidic conditions. In this study, we identified and characterized the key compounds involved in the antioxidant activity of this fraction. We first isolated two MAAs, palythine, and porphyra-334, from the fraction and evaluated the activities of the two MAAs when heated. MAAs possess absorption maxima at around 330 nm, while the heated MAAs lost this absorption. The heated MAAs showed a high ABTS radical scavenging activity at pH 5.8–8.0. We then determined the structure of heated palythine via ESI-MS and NMR analyses and speculated about the putative antioxidant mechanism. Finally, a suitable production condition of the heated compounds was determined at 120 °C for 30 min at pH 8.0. We revealed compounds from red algae with antioxidant activities at a wide range of pH values, and this information will be useful for the functional processing of food.     

Isolation of Molecular Markers Linked to the <Emphasis Type="Italic">Cry</Emphasis> Locus Conferring Resistance to <Emphasis Type="Italic">Cucumber mosaic cucumovirus </Emphasis>Infection in Cowpea

We previously demonstrated that cowpea [Vigna unguiculata (L.) Walp.] cultivar Kurodane-Sanjaku contains the Cry gene, which confers resistance against Cucumber mosaic cucumovirus infection. In this paper, randomly amplified polymorphic DNA (RAPD) analysis was carried out to tag the Cry locus. Bulked segregant analysis for RAPD resulted in many polymorphisms in amplified DNA patterns. Candidates were further screened using parental and/or F₂ cowpea DNAs. As a result, we obtained three RAPD markers, D13/E14-350, WA3-850 and OPE3-500, flanking the Cry locus. In addition, we amplified cowpea sequences coding for the putative nucleotide-binding site (NBS). Degenerate primers based on NBS sequences of tobacco N and Arabidopsis RPS2 disease resistance genes were used for polymerase chain reaction, and resultant products were cloned and sequenced. Among eight independent clones, cowpea resistance gene analog (CRGA) 5 showed a distinct polymorphism when used as a probe for restriction fragment length polymorphism analysis against the susceptible cowpea cultivar PI 189375 and a near-isogenic line for the Cry. Linkage analyses of these molecular markers showed that genetic distances of CRGA5, D13/E14-350, WA3-850 and OPE3-500 to the Cry locus were 0.7, 5.2, 11.5 and 24.5 cM, respectively. Received 16 December 1999/ Accepted in revised form 22 March 2000     

Effect of dietary taurine and lipid contents on conjugated bile acid composition and growth performance of juvenile Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

ABSTRACT:   The effects of dietary taurine levels and lipid contents on the conjugated bile acid composition and growth performance of juvenile Japanese flounder Paralichthys olivaceus were investigated. Six types of diet (three different levels of taurine at two different levels of lipid) were fed to juveniles (average body weight, 0.04 g). Fishmeal that was washed with 70% ethanol to remove taurine was used as the sole protein source. Feeding experiments were carried out at 20°C for 6 weeks. At the end of the experiments, fish were weighed and analyzed for free amino acids in the body and the composition of the conjugated bile acids. The body weight and percent weight gain of the juveniles were improved by the dietary taurine supplementation. The taurine contents of the whole body and tissues increased with the increase of the dietary taurine contents. The conjugated bile acids in the gall bladder consisted of taurocholic acid and taurochenodeoxycholic acid, which increased with the increase of the dietary taurine level. Taurocholic acid accounted for more than 95% of the total conjugated bile acids. This indicates that taurine is the sole amino acid to conjugate bile acid in Japanese flounder.