In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells

To verify the importance of somatic cells upon in vitro embryo development, in vitro-matured (IVM) and -fertilized (IVF) bovine oocytes were cultured in TCM 199 supplemented with estrous cow serum (10% v/v) and 0.25 mM sodium pyruvate (ECSTCM) under the following treatments: 1) ECSTCM alone; 2) together with bovine oviduct epithelial cells (BOEC); 3) with cumulus cells (CC); 4) in fresh BOEC conditioned ECSTCM; or 5) in frozen-thawed BOEC conditioned ECSTCM. Culturing zygotes encased in cumulus cells significantly reduced the cleavage rate (P<0.05). There was no difference between culture systems in the proportions of embryo development through the 8-cell stage (P=0.42) up to the morula/blastocyst stages (P=0.50) at Day 7 post insemination. However, co-culture with BOEC yielded the highest percentage (21.2% of zygotes; P<0.05) of quality Grade-1 and Grade-2 embryos with the number of blastomeres per embryo (114.4) comparable to that of 7-day-old in vivo-developed embryos of similar grades (102.5), and higher (P<0.05) than those of the other treatments. The ratio of blastocysts to total morulae/blastocysts obtained from frozen-thawed conditioned medium was lower (P<0.05) than that from ECSTCM or after co-culture with BOEC at Day 7 post insemination. On average, 7.5 to 17.5% of the zygotes developed to blastocyst, expanded blastocyst and hatched blastocyst stages by Day 10 post insemination, depending upon the culture system. The difference between treatments, however, was not significant (P=0.68). The results indicate that chronological development up to hatching of bovine IVM-IVF embryos is not favored by somatic cells; however, the presence of viable oviduct epithelial cells in culture significantly improves the quality of 7-day-old embryos.     

Eicosanoid production in experimental alcoholic liver disease is related to vitamin E levels and lipid peroxidation

We investigated the association between vitamin E, lipid peroxidation and eicosanoid production in experimental alcoholic liver injury. We used the intragastric feeding rat model in which animals were fed corn oil and ethanol (CO+E) and corn oil and dextrose (CO+D) for 2 and 4 week periods. At sacrifice, we measured plasma levels of alpha-tocopherol, 8-isoprostane, thromboxane B₂(TXB₂) and 6-ketoprostaglandin F₁(6-KetoPGF₁). Animals fed CO+E had significantly lower concentrations of -tocopherol and higher concentrations of 8 isoprostane at both 2 and 4 weeks. a significant inverse correlation was seen between -tocopherol concentrations and the TXB₂: PGF₁ ratio (r=0.72, p<0.01). A positive correlation was seen between the TXB₂: PGF₁ ratio and 8 isoprostane levels (r=0.84, p<0.001). These results suggest that vitamin E depletion and enhanced lipid peroxidation may affect eicosanoid metabolism in experimental alcoholic liver disease in such a way so as to increase the thromboxane to prostacyclin ration.Abbreviations PGH₂ Prostaglandin H₂ - PGL₂ Prostacyclin - CO+E Corn Oil and Ethanol - CO+D Corn Oil and Dextrose - TXB₂ Thromboxane B₂ - TXA₂ Thromboxane A₂     

Effect of Growth Factors on Bovine Blastocyst Development in a Serum-Free Medium

To investigate the effect of growth factors on pre-implantation development, bovine zygotes, produced by in vitro fertilization (IVF) of in vitro-matured (IVM) oocytes, were cultured in a serum-free medium to which the following growth factors were added one at a time: epidermal growth factor (EGF), acidic fibroblast growth factor (a-FGF), insulin-like growth factor-II (IGF-II), platelet-derived growth factor from human platelets (PDGF), and platelet-derived growth factor-AB, human, recombinant (PDGF-AB). All growth factors were added at a dose of either 10 or 50 ng/ml, except PDGF which was added at a dose of either 5 or 15 ng/ml. The control medium was TCM 199 supplemented with sodium pyruvate (0.25 mmol/1), BSA (10 mg/ml), insulin (5 μg/ml), transferrin (5 μg/ml), and sodium selenite (5 ng/ml). Embryos were cultured for 8 days (day of insemination = Day 0). The mean percentages of first cleavage on Day 2 varied from 67% to 86% and the differences between the 2 doses, or between the control and growth factor- treated groups were not significant (p≥0.13). The effects of the two doses on subsequent development up to the blastocyst stage did not differ either (p≥0.12). There was no stimulatory effect of any of the used exogenous growth factors on embryo development up to the morula or blastocyst stage on Day 7, or blastocyst stage on Day 8. Moreover, medium supplemented with PDGF had fewer blastocysts than the control (p≤0.03). The results indicate that growth factor supplementation may not necessarily increase the yield of blastocysts from bovine IVM-IVF oocytes in a serum-free medium.     

Spirulina ameliorates arsenic induced reproductive toxicity in male rats

Spirulina (Spirulina platensis), has numerous health benefits including antioxidant, immunomodulatory, and anti-inflammatory activities, works against heavy metal toxicity, and is often used as a food supplement in human, animals, birds and fishes. This study aimed to evaluate the protective ability of the dietary spirulina against the toxic effects of inorganic arsenic (iAs) on male reproductive parameters in rats. Seventy-two mature Long-Evans male rats, dividing into six groups (T0, T1, T2, T3, T4 and T5) (12 rats/group) were included in this study. The T3, T4 and T5 group rats were treated with three consecutive doses (1.0 g, 1.5 g and 2.0 g/kg feed) of spirulina in feed along with 3.0 mg NaAsO₂/kg body weight (BW) in drinking water (DW) daily for 90 days. Each rat of group T1 received NaAsO₂ (3.0 mg/kg BW) in DW, and those of T2 group were fed with spirulina (2.0 g/kg feed) daily for 90 days. The rats of group T0 served as the control with normal feed and water. Total arsenic (tAs) contents, reproductive parameters (testicular weight, sperm motility and morphology), and histological changes in the testicles were evaluated in these rats. Arsenic dosing significantly (p=0.003, Kruskal-Wallis test) increased the tAs contents in the testicles, decreased testes weight, sperm morphology and motility compared to the controls. The effect of arsenic dosing was also evidenced by the histological changes like decreased germinal layers in the seminiferous tubules of the treated rats. Moreover, dietary spirulina (2.0 g/kg feed) supplementation significantly (p=0.011, Kruskal-Wallis test) lowered tAs contents in testicles and increases testes weights, sperm motility and morphology. Therefore, spirulina can be used as an effective dietary supplement to ameliorate the adverse effects of arsenic induced reproductive toxicities. However, further study is required to elucidate the underlying molecular mechanisms of reduction of arsenic induced reproductive toxicity by spirulina.     

Complex combination of seed dormancy and seedling development determine emergence of Viburnum tinus (Caprifoliaceae)

BACKGROUND AND AIMS: The shrub Viburnum tinus is widely distributed in mattoral vegetation of the Mediterranean basin. The purpose of the present study was to classify the seed dormancy type and examine the requirements for embryo growth, root protrusion and shoot emergence. METHODS: Overwintered fruits were collected in western Spain in April 2001 and prepared in three ways: entire pericarp was removed, exocarp and mesocarp were removed or fruits were left intact. Fruits treated in these three ways were subjected to artificial annual temperature cycles or to constant temperature regimes for 1.5 years. KEY RESULTS: Removal of exocarp and mesocarp was necessary for embryo growth and germination. High temperature favoured dormancy alleviation and embryo growth, intermediate to low temperatures favoured root protrusion, and intermediate temperature shoot emergence. There was substantial germination at constant temperature regimes, indicating an overlap between temperature intervals suitable for the different stages of embryo and seedling development. Functionally, V. tinus has the same root and shoot emergence pattern that is described for other Viburnum species considered to have epicotyl dormancy. However, the requirement for high and low temperatures for radicle protrusion and epicotyl emergence, respectively, was missing in V. tinus; these characters are the foundation for the epicotyl dormancy classification. CONCLUSIONS: It is concluded that V. tinus does not have epicotyl dormancy. Instead, there is a combination of a weak morphophysiological dormancy and a slow germination process, where different temperatures during an annual cycle favour different development stages. The present study suggests that the first complete seedlings would emerge in the field 1.5 years after fruit maturation in October, i.e. seed dispersal during winter, embryo growth during the first summer, root protrusion and establishment during the second autumn and winter, and cotyledon emergence during the second spring.     

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.     

Effects of exogenous inositol hexakisphosphate (InsP(6)) on the levels of InsP(6) and of inositol trisphosphate (InsP(3)) in malignant cells,tissues and biological fluids

InsP(6) is abundant in cereals and legumes. InsP(6) and lower inositol phosphates, in particular InsP(3), participate in important intracellular processes. In addition, InsP(6) possess significant health benefits, such as anti-cancer effect, kidney stones prevention, lowering serum cholesterol. Because of the insensitivity of existing methods for determination of non-radiolabeled inositol phosphates, little is known about the natural occurrence, much less on the concentrations of InsP(6) and InsP(3) in biological samples. Using gas chromatography-mass detection analysis of HPLC chromatographic fractions, we report a measurement of unlabeled total InsP(3) and InsP(6) (a) as they occur within cells culture, tissues, and plasma, and (b) their changes depending on the presence of exogenous InsP(6). When rats were fed on a purified diet in which InsP(6) was undetectable (AIN-76A) the levels of InsP(6) in brain were 3.35 +/- 0.57 (SE) micromol.kg(-1) and in plasma 0.023 +/- 0.008 (SE) micromol.l(-1). The presence of InsP(6) in diet dramatically influenced its levels in brain and in plasma. When rats were given an InsP(6)-sufficient diet (AIN-76A + 1% InsP(6)), the levels of InsP(6) were about 100-fold higher in brain tissues (36.8 +/- 1.8 (SE)) than in plasma (0.29 +/- 0.02 (SE)); InsP(6) concentrations were 8.5-fold higher than total InsP(3) concentrations in either plasma (0.033 +/- 0.012 (SE)) and brain (4.21 +/- 0.55 (SE)). When animals were given an InsP(6)-poor diet (AIN-76A only), there was a 90% decrease in InsP(6) content in both brain tissue and plasma (p < 0.001); however, there was no change in the level of total InsP(3). In non-stimulated malignant cells (MDA-MB 231 and K562) the InsP(6) contents were 16.2 +/- 9.1 (SE) micromol.kg(-1) for MDA-MB 231 cells and 15.6 +/- 2.7 (SE) for K 562 cells. These values were around 3-fold higher than those of InsP(3) (4.8 +/- 0.5 micromol.kg(-1) and 6.9 +/- 0.1 (SE) for MDA-MB 231 and K562 cells respectively). Treatment of malignant cells with InsP(6) resulted in a 2-fold increase in the intracellular concentrations of total InsP(3) (9.5 +/- 1.3 (SE) and 10.8 +/- 1.0 (SE) micromol.kg(-1) for MDA-MB 231 and K562 cells respectively, p < 0.05), without changes in InsP(6) levels. These results indicate that exogenous InsP(6) directly affects its physiological levels in plasma and brain of normal rats without changes on the total InsP(3) levels. Although a similar fluctuation of InsP(6) concentration was not seen in human malignant cell lines following InsP(6) treatment, an increased intracellular levels of total InsP(3) was clearly observed.     

Seed germination ecophysiology of the Asian species Osmorhiza aristata (Apiaceae): comparison with its North American congeners and implications for evolution of types of dormancy

Osmorhiza aristata is an herbaceous perennial that grows primarily in Japan, through southern China, to the Himalayas. It closely resembles the eastern North American species O. claytonii and O. longistylis, and, together, the three species are an example of the well-known North American-Asian pattern of disjunction. Requirements for dormancy break and embryo growth were determined for seeds of O. aristata collected in Japan during the summers of 1998-2000. Embryos in fresh seeds were ca. 0.5 mm long, and they had to grow to 9 mm before the radicle emerged from the mericarp. Embryo growth and germination occurred during cold stratification at 5°C, the optimum temperature for germination. Gibberellic acid did not substitute for cold stratification. Thus, O. aristata seeds have deep complex morphophysiological dormancy (MPD). The type of MPD in O. aristata is similar to that in two western North American congeners but different from that in eastern North American congeners (nondeep complex MPD). Mapping the types of MPD onto a phylogeny of the genus suggests that nondeep complex MPD is derived from deep complex MPD. Although eastern North American-Asian disjuncts often exhibit morphological stasis, the taxa may differ greatly in physiological traits, such as seed dormancy.     

Biodiversity and distribution of fungi associated with decomposing Nypa fruticans

Fungi associated with the decomposition of Nypa fruticans in Malaysia are under investigation. Forty-one fungi have been identified including 35 ascomycetes, four mitosporic fungi and two basidiomycetes. The distribution of intertidal fungi on palm structures including leaves, leaf veins, rachides, petiole bases, and inflorescences, and fungi on terrestrial parts have also been examined. No fungi were found on the leaf material, although several fungi were found on the leaf midribs, and possible reasons for this are given. Very few taxa developed on the inflorescences, but those that were present were abundant. The greatest density of fruiting structures occurred on the rachides, and the greatest diversity of fungal species occurred on the petiole bases. The terrestrial fungi differed from the intertidal fungi, although Linocarpon nipae occurred in both habitats. Reasons for the differences in fungal numbers and diversity on the various palm parts are discussed. The diversity of fungi at Morib mangrove was low when compared to previous studies on fungi on Nypa palm at Kampong Api Api in Brunei and in this study at Kuala Selangor mangrove in Malaysia.     

The effect of seaweed composite flour on the textural properties of dough and bread

Seaweeds as food and seaweed-derived food flavors, colors, and nutrients are attracting considerable commercial attention. In the baking industries, hydrocolloids are of increasing importance as bread making improvers, where their use aims to improve dough handling properties, increase the quality of fresh bread, and extend the shelf life of stored bread. Seaweeds contain a significant amount of soluble polysaccharides and have the potential function as a source of dietary fiber. In this study, red seaweed (Kappaphycus alvarezii) powder was incorporated (2–8 %) with wheat flour and used to produce bread. The effect of seaweed composite flour on dough rheological properties and the quality of bread was investigated using various techniques. Farinograph tests were applied to determine the effect of seaweed powder on the rheological properties of wheat flour dough, while texture profile analysis (TPA) was used to measure the textural properties of dough as well as the final product. The results showed that the additions of seaweed powder (2–8 %) increased the water absorption of the dough. TPA results showed that the addition of seaweed powder decreased stickiness properties. Bread produced with seaweed composite flour showed higher values of firmness.