Three-dimensional live imaging of bovine embryos by optical coherence tomography

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.     

Real-time PCR for differential determination of the tomato wilt fungus, <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>, and its races

Five primer/probe sets to identify the tomato wilt pathogen, Fusarium oxysporum f. sp. lycopersici (FOL), and its three races selectively were designed based on the rDNA-intergenic spacer and avirulence genes. Real-time PCR using genomic DNA from mycelia and soil DNA with the primer/probe sets allowed the successful identification of FOL and its races.     

Practical method combining loop-mediated isothermal amplification and bait trap to detect <Emphasis Type="Italic">Pythium helicoides</Emphasis> from hydroponic culture solutions

Two detection methods combining loop-mediated isothermal amplification (LAMP) and a bait trap were developed to detect Pythium helicoides in greenhouses containing roses, miniature roses, and poinsettias in hydroponic culture systems. In “Bait-LAMP”, a crude extract derived from perilla seeds as the bait was used in the LAMP reaction, whereas in the “Bait culture-LAMP”, a crude extract of mycelia grown out from perilla seeds onto Pythium-selective medium served as the bait. The two methods are simple and rapid for practical monitoring of P. helicoides in hydroponic culture systems.     

The effect of ovary storage and in vitro maturation on mRNA levels in bovine oocytes; a possible impact of maternal ATP1A1 on blastocyst development in slaughterhouse-derived oocytes

Since BSE testing of slaughtered cattle is obligatory in Japan, storage of ovaries at 15-20 C overnight in phosphate buffered saline has become a routine protocol in in vitro production (IVP) of cattle embryos. Ovary storage is known to reduce developmental competence of oocytes; however, its effects on oocyte gene expression have not been clarified yet. This study compared oocytes collected from stored slaughterhouse-derived ovaries with those collected by Ovum Pick-Up (OPU) in terms of the expression of 20 selected genes to determine if ovary storage affects cellular processes at the molecular level. Expression of mRNA in oocytes was assayed before and after in vitro maturation (IVM) by real-time quantitative PCR. Maternal mRNA levels of genes were investigated in 2-cell stage embryos obtained from slaughterhouse oocytes to assess their roles for blastocyst formation. In immature OPU oocytes, genes related to metabolism (GAPDH), transporters (GLUT8, ATP1A1) and stress resistance protein (HSP70) showed significantly higher expression compared with oocytes derived from stored ovaries. During IVM, the expression of GDF9, GLUT8, CTNNB1 and PMSB1 was significantly decreased irrespective of oocyte source. Two-cell stage embryos cleaving at 22-25 h after in vitro fertilization (IVF) showed a significantly higher blastocyst formation rate and ATP1A1 gene expression level compared with those cleaving at 27-30 h after IVF. Our results reveal that storage of ovaries alters mRNA levels in oocytes. Correlation of Na/K ATPase ATP1A1 expression in IVP embryos at the 2-cell and 8-cell stages with their developmental ability to the blastocyst stage may suggest the importance of maternal mRNA of this gene during blastulation in embryos derived from slaughterhouse oocytes.     

A 39 kDa protein mediates adhesion of avian Pasteurella multocida to chicken embryo fibroblast cells

To clarify the role of avian Pasteurella multocida capsule in pathogenesis, adhesion of capsulated strains P-1059, X-73 and Pm-18, and noncapsulated strains P-1059B, Pm-1 and Pm-3 to chicken embryo fibroblast (CEF) cells was compared. Number of adherent organisms of the capsulated strains to CEF cells were approximately three times as much as noncapsulated strains indicating that adhesive properties were enhanced by the presence of bacterial capsule. Pretreatments of the bacterial cells with heat, trypsin, or with antiserum caused a marked decrease in adhesion of capsulated strain P-1059 and its noncapsulated variant P-1059B. However, depolymerization of capsular hyaluronic acid with high dose of hyaluronidase enhanced adhesion of these strains. Combined treatments of the bacterial cells with both hyaluronidase and trypsin significantly (P < 0.05) inhibited the adherence of strain P-1059 as compared to the treatment only with trypsin, but strain P-1059B was not affected. SDS-PAGE profiles of crude capsular extract (CCE) prepared from capsulated strain P-1059 and its noncapsulated variant P-1059B grown on dextrose starch agar (DSA) plates by heating at 56 degrees C in a 2.5% NaCl solution demonstrated eight protein bands of 28, 34, 36, 39, 52, 56, 63 and 93 kDa. The 28, 34 and 36 kDa proteins were commonly major for both strains, and the 39 kDa protein was major only for strain P-1059 but poor in strain P-1059B. Outer membrane protein (OMP) profiles were identical with a major protein at 34 kDa and four minor proteins between the two strains. The adhesion of strain P-1059 and strain P-1059B to CEF cells was inhibited significantly (P < 0.01) by treatment with rabbit antisera against P-1059, P-1059B, CCE or 39 kDa protein of strain P-1059 as compared to the treatment with either PBS or with normal rabbit serum. These results indicated that an antigenic 39 kDa protein in the capsule may be responsible for adhesion of avian P. multocida type A strains to CEF cells as a virulence factor.     

Kinetics of endotoxin concentration and tumor necrosis factor-alpha,interleukin-1beta,and interleukin-6 activities in the systemic and portal circulation during small intestinal ischemia and reperfusion in dogs

OBJECTIVE: To determine whether small intestinal ischemia and reperfusion induces bacterial translocation and proinflammatory cytokine response in either the systemic or portal circulation in dogs. ANIMALS: 17 healthy adult Beagles. PROCEDURE: The superior mesenteric artery (SMA) was occluded for 0 (group-3 dogs), 30 (group-1 dogs), or 60 (group-2 dogs) minutes, followed by reperfusion for 180 minutes; serum lactate and endotoxin concentrations and tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 activities in the systemic and portal circulation and intramucosal pH were measured at various time points. RESULTS: In group-2 dogs, TNFalpha activity was found to be significantly increased in the portal circulation, peaking at 60 minutes of reperfusion; TNF-alpha activity, in the systemic circulation, gradually increased from 60 minutes of reperfusion to the end of the experiment; however, the increase was not significant. In group-1 and -2 dogs, IL-6 activities significantly and gradually increased in the systemic and portal circulation during the reperfusion phase, and the magnitude of these increases was dependent on the duration of the ischemic phase. There were no significant changes in IL-1beta activity or endotoxin concentration in any dog group. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the our study indicate that intestinal ischemia and reperfusion leads to significant increases of the circulating TNF-alpha and IL-6 activities, depending on the duration of the ischemia phase, in the absence of detectable endotoxin in the circulation. This finding suggests that intestinal ischemia and reperfusion induces a systemic proinflammatory cytokine response in dogs.     

Gonadosomatic index and testis morphology of common carp (Cyprinus carpio) in rivers contaminated with estrogenic chemicals

To study the effect of estrogenic chemicals on fish, the gonadosomatic index (GSI = [testis weight/body weight] x 100) and testis histology of mature common carp (Cyprinus carpio) from 2 contaminated sites (Ishizu and Wada rivers, Osaka) and a control site were examined between June 1998 and March 2001. The concentration of nonylphenol, bisphenol A and 17beta-estradiol in the Ishizu river was 3-4 times higher than in the Wada river. In the pre-breeding and breeding seasons, there were no significant differences in body weight among carp from the 3 sites, the body weight of Ishizu river carp being significantly lower (p<0.05) than that of Wada river fish only in the post-breeding season. The GSI and testis weight in fish from the Ishizu river were significantly lower (p<0.05) than in control fish during all phases of gonadal cycle and lower than in Wada river fish in the pre-breeding and post-breeding season. No histological abnormalities were found in the testes of the males examined. Histological observation of the testes revealed a delay in the onset of spermatogenesis in fish from the Ishizu river compared with those from the other sites. These results clearly imply that the estrogenic chemicals in the Ishizu river adversely affect the testis development of the fish.     

Physicochemical Properties and Molecular Structures of Starches from Millet (Pennisetum typhoides) and Sorghum (Sorghum bicolor L. Moench) Cultivars in Nigeria

Some physicochemical properties and molecular structures of starches from millet (Pennisetum typhoides, Doro and Gero) and sorghum (Sorghum bicolor, red and white) in Nigeria were examined. Starch granules of millet and sorghum were 3–14 μm and 4–26 μm in diameter, respectively. Millet cultivars had similar peak viscosities (204–205 RVU) on pasting, while sorghum showed similar minimum viscosities (155–156 RVU). The actual amylose content (%) calculated from iodine affinity (IA, g/100 g) was 20.1 and 21.4 for sorghum and 21.3 for millet. The IA of amylopectin was high (1.27–1.42) and its average chain lengths were 20–21 with β‐amylolysis limit of 56%. Amylopectins showed a polymodal molecular weight distribution on a molar basis. The distributions differed among the samples with a higher amount of larger molecules in Doro and red sorghum. Weight‐ and molar‐based distributions of debranched amylopectins on HPSEC were polymodal with weight‐based distribution showing presence of long chains. Peak DP values for A+B₁ and B₂+B₃ chain fractions were 13–16 and 42–43, respectively. The (A+B₁)/(B₂+B₃) ratio on molar basis (9.0–11.5) was similar to maize and rice amylopectins. Peak DP on molar‐based distribution for white sorghum and millet amyloses were similar (490–540) and the DP_n range was narrow (1,060–1,300), but weight‐based distribution profiles differed. The average chain lengths were 260–270 with 3.9–4.8 chains per molecule.