The effect of sperm preparation and co-incubation time on in vitro fertilization of Bos indicus oocytes.

The objective of the present study was to evaluate the effect of various methods of sperm selection and various sperm-oocyte co-incubation times on in vitro fertilization (IVF) of zebu (Bos indicus) oocytes. Frozen semen from one ejaculate of a single bull was used for all treatments and replicates. After thawed, sperm was subjected to one of the three treatments: 45 and 90% discontinuous Percoll gradient, swim-up and washing by centrifugation. In all treatments, the spermatozoa were incubated with in vitro matured oocytes for 3, 6, 12 and 18h. After co-incubation oocytes were transferred to the culture medium and culture for 44h, when the cleavage was evaluated. The uncleavaged oocytes were fixed and stained to determine penetration, pronucleus formation and polyspermy. The sperm selection method did not influence (P<0.05) polyspermy, pronucleus formation, penetration and cleavage rates. No interaction between method of selection and sperm-oocyte co-incubation time was observed (P>0.05). However, sperm-oocyte co-incubation time affected fertilization. The lower penetration (26.5%) and cleavage rates (13.1%) were obtained at 3-h period. The penetration and cleavage percentages increased (P<0.05) progressively at 6h (63.3 and 54.4%) and 12h (77.6 and 67.6%). No differences (P>0.05) were observed between 12 and 18h of incubation for penetration and cleavage rates. The incidence of polyspermy and pronucleus formation was similar (P>0.05) for all time points. It is concluded that the methods used in this study for sperm selection do not affect fertilization; therefore, they all can be used for bovine IVF. In addition, regardless the method used better fertilization results were obtained when sperm and oocytes were co-incubated for 12h, and the prolongation of that time for up to 18h had no detrimental effect on fertilization.     

The Growth Response of the Stems of Genetically Modified Tobacco Plants (Nicotiana tabacum'Samsun') to Flexural Stimulation

Genetically modified tobacco plants (Nicotiana tabacum‘Samsun’)with antisense cinnamyl alcohol dehydrogenase DNA, produce secondaryxylem of a reduced tensile stiffness. These plants were grownalongside control plants. The stems of the plants were flexedor protected from flexing over a period of several weeks. Thetensile moduli and second moments of areas of the differenttissues inside the stems were measured and used to calculatethe bending stiffness of the plants. In tobacco, the cylinderof xylem was found to be the most important tissue in determiningthe bending stiffness of the plants. The thickness of the xylemtissue cylinder increased when plants were subjected to flexuralstimulation. This increased the bending stiffness of the stems.The response to mechanical stimulation was found to be correlatedwith tissue strain and the genetically modified plants wereable to exactly compensate for the reduced modulus of theirxylem tissue by increasing the thickness of the xylem tissuecylinder more than in control plants.Copyright 1999 Annals ofBotany Company. Tobacco plants, stem bending, xylem tissue, second moment of area, thigmomorphogenesis, mechanical strain.     

Levels of genetic diversity at different stages of the domestication cycle of interior spruce in British Columbia

Concerns over the reductionist nature of the domestication of forest-tree species focus on the possibility of potential genetic erosion during this process. To address these concerns, genetic diversity assessments in a breeding zone the Province of British Columbia “interior” spruce (Picea glauca×engelmanni) program was conducted using allozyme markers. Genetic-variation comparisons were made between natural and production (seed orchard) populations as well as seed and seedling crops produced from the same breeding zone’s seed orchard. The natural population sample consisted of a total of 360 trees representing three stands within each of three watersheds present in the Shuswap-Adams low-elevation zone of interior British Columbia. Small amounts of genetic differentiation were observed among the nine natural populations (4%) and this was attributable to extensive gene flow Consequently, the sum of these nine populations was considered as a baseline for the genetic variation present in the breeding zone. The comparisons between the seed orchard and the breeding zone produced a similar percentage of polymorphic loci while the expected hetrozygosity (0.207 vs 0.210) and the average number of alleles per locus (2.7 vs 2.4) were slightly lower in the seed orchard. A total of seven natural populations’ rare alleles were not present in the orchard population, while one allele was unique to the orchard. The %P increased to 70.6% in the seedlot, but dropped to the natural populations level (64.7%) in the plantation. The observed increase in %P was a result of pollen contamination in the orchard. It is suspected that the reduction in the plantation was caused by an unintentional selection in the nursery. Simulated roguing in the orchard did not drastically reduce even if up to 50% of the orchard’s clones were rogued. However, roguing was associated with a reduction in the average number of alleles per locus (i.e., sampling effect). Received: 2 January 1996 / Accepted: 24 May 1996     

Detailed flow patterns in the nasal cavity.

The human nasal cavity filters and conditions inspired air while providing olfactory function. Detailed experimental study of nasal airflow patterns has been limited because of the complex geometry of the nasal cavity. In this work, particle image velocimetry was used to determine two-dimensional instantaneous velocity vector fields in parallel planes throughout a model of the nasal cavity that was subjected to a nonoscillatory flow rate of 125 ml/s. The model, which was fabricated from 26 computed tomography scans by using rapid prototyping techniques, is a scaled replica of a human right nasal cavity. The resulting vector plots show that the flow is laminar and regions of highest velocity are in the nasal valve and in the inferior airway. The relatively low flow in the olfactory region appears to protect the olfactory bulb from particulate pollutants. Low flows were also observed in the nasal meatuses, whose primary function has been the subject of debate. Comparison of sequentially recorded data suggests a steady flow.     

Castilleja chambersii (Scrophulariaceae), a new rare species from the northern Coast Range of Oregon

Castilleja chambersii is described from several collections made in the Coast Range of southwestern Clatsop County, Oregon. The new species is a member of subgenusCastilleja and is most closely related toC. parviflora andC. rupicola. This rare species is known from only three small and geographically restricted populations. Two new meiotic chromosome counts ofn=12 are reported for the new species.     

The relation between hip impact velocity and hip impact force differs between sideways fall techniques.

Fall techniques that reduce fall severity may decrease the risk of hip fractures. A fundamental variable for fall severity is impact force, but impact velocity is also used. The purpose of the study was to determine whether impact velocity is valid to determine differences in fall severity between different techniques. Five young adults with martial arts (MA) experience performed sideways falls from kneeling height using three techniques: Block with arm (Block) and MA techniques with and without use of the arm to break the fall. In addition, one subject also performed MA falls from standing height. Linear regression analysis showed a moderate relation between hip impact velocity and force, which was depended on technique. In falls with comparable impact velocities, forces in MA falls were lower than forces in Block falls. Hence, differences in impact force could not be predicted by velocity. In conclusion, hip impact velocity may be useful to make an approximate prediction of impact force within fall techniques. However, to determine differences between techniques it was not always a valid predictor. When direct impact force measurements are not possible, methods combining impact velocity with energy estimates before and after impact might be more valid.     

Haploid plant regeneration from unpollinated ovule cultures of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis> (L. f.) Cass.)

Haploid plants were regenerated in vitro from unpollinated ovules of niger (Guizotia abyssinica (L. f.) (Cass.) on Murashige and Skoog nutrient medium (MS) supplemented with 10 μM naphthaleneacetic acid or 10 μM NAA + 1.5 μM kinetin and 30 g/l sucrose. Gamborg (B5) medium was the best for plant regeneration (in comparison with MS, Nitsch and Nitsch (NN), and Chu (N6) media) from cultured ovules, and 6.66 and 7.33 ovules of JNC-6 and Ootacamund cultivars were involved in direct plant regeneration on this medium. Matured ovules (ovules collected one day before anthesis or on the day of anthesis) only responded to cultural regimes and involved in direct plantlet development. Cytological preparation of root tips and chloroplast counts in the guard cells of leaf stomata of regenerated plants confirmed their haploid nature. This text was submitted by the authors in English.     

Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

Promotion by phloroglucinol of micropropagation of Minuartia valentina, an endangered and endemic Spanish plant

Tissue culture techniques for the propagation and conservation of endemic or threatened plants can be used to complement the methods usually applied in ex situ conservation. Thus, Minuartia valentina (Caryophyllaceae), an endangered plant species endemic to the Valencia Community (Eastern Spain), was successfully regenerated through shoot proliferation from wild plants growing in their natural area. Nodal segments, 10~mm long, were cut from rametes of adult material, sterilised and established in vitro. Equally successful shoot multiplication was achieved on Murashige and Skoog (MS) medium with 80 mg l-1 phloroglucinol in combination with either 1 mg l-1 6-benzylaminopurine or 1 mg l-1 kinetin. Excised shoots were rooted in MS medium supplemented with an auxin (indole acetic acid, indole-3-butyric acid, or napththalene acetic acid). Shoots rooted well (96–100%) within three weeks in all auxin treatments. However, the use of napththalene acetic acid was discarded because this auxin delayed root differentiation, and induced adventitious root malformation. Rooted plantlets were transferred to pots and 85% of them acclimatized successfully four weeks after transfer to greenhouse conditions, where they exhibited normal morphology and growth.