Morphology of Dromedary Camel Oocytes and their Ability to Spontaneous and Chemical Parthenogenetic Activation

The present work was conducted to examine (1) the morphology of dromedary cumulus‐oocytes complexes (COCs), (2) to study the incidence of spontaneous development of oocytes in vivo and (3) to assess the ability of in vitro matured dromedary oocytes to chemical parthenogenetic activation compared with in vitro fertilized (IVF) oocytes. COCs were recovered from dromedary ovaries classified according to their morphology into six categories. Oocyte diameter was measured using eye piece micrometer. For chemical activation, COCs with at least three layers of cumulus‐cells were in vitro matured (IVM) in TCM 199 + 10 μg/ml FSH + 10 IU hCG/ml + 10% FCS + 50 μg/ml gentamycin. COCs were incubated for 40 h at 38.5°C under 5% CO₂ in humidified air. After IVM, matured oocytes with first polar body (first Pb) were divided into two groups. Group 1: activated in 7% ethanol (E) for 5 min followed by culture in 2 mM 6‐dimethylaminopurin (6‐DMAP, E D, subgroup 1) or 10 μg/ml cycloheximide (CHX, E CHX, subgroup 2) for 3.5 h at 38.5°C under 5% CO₂. In group 2, oocytes were activated using 50 μM Ca A23187 (Ca A) for 5 min followed by culture in 2 mM 6‐DMAP (Ca D, subgroup 3) or 10 μg/ml CHX(Ca CHX, subgroup 4) for 3.5 h at 38.5°C under 5% CO₂. For control group, IVM oocytes were fertilized using frozen‐thawed camel spermatozoa separated by swim‐up method then suspended in Fert‐TALP medium supplemented with 6 mg/ml BSA (FAF) + 10 μg/ml heparin. In all groups, oocytes were in vitro cultured in SOFaa medium + 5% FCS and 5 μg/ml insulin + 50 μg/ml gentamycin. Cleavage rate and embryo development were checked on Days 2, 5 and 8. An average of 11.3 ± 0.3 COCs were recovered/dromedary ovary. Categories 1 and 2 represented 33.1% and 34.8%, respectively, and were significantly higher (p < 0.01) than the other categories (19.1, 9.2 and 2.6% for categories 3–5, respectively). Category 6 (embryo‐like structures) represented 1.2% of the recovered oocytes, staining of these embryo‐like structures with orcien dye indicated the presence of divided cells with condensed nuclei. Dromedary oocytes averaged 166.2 ± 2.6 μm in diameter with black cytoplasm. Chemical activation of IVM dromedary oocyte with first Pb in 7% ethanol or 50 μM Ca A followed by culture in 2 mM 6‐DMAP showed significantly higher (p < 0.01) cleavage and developmental rates to the morula stage than oocytes activated using 7% ethanol or 50 μM Ca A followed by 10 μg/ml CHX or in vitro fertilized control group. Higher (p < 0.01) proportion of oocytes sequentially cultured in 10 μg/ml CHX or that in vitro fertilized were arrested at the 2–4‐cell stage compared with that cultured in 6‐DMAP.     

Dormancy and viability of Phalaris minor seed in a rice–wheat cropping system

Seed placement, soil temperature and soil moisture content influenced the process of after-ripening in Phalaris minor seeds. Seeds of P. minor collected from the soil just after wheat harvesting exhibited higher germination than seeds from P. minor threshed directly. There was a pronounced impact of periodic inhabitation of seed into the soil on germination after its dispersal. Germination was strongly inhibited when the seed was kept in soil at more than field capacity (FC) or in water. Maximum germination of seed incubated in soil at FC occurred at 30°C while a temperature of 40°C favoured after-ripening of seed when mixed with dry soil or kept dry without any medium. Release from conditional dormancy was quicker in the seed retrieved from the soil kept at 20°C than at 10°C. Seed release from conditional dormancy and germination increased with a rise in temperature from 30 to 40°C when the seed was retrieved from incubation in soil at FC for 70 days. The seed kept immersed in water was least responsive to a rise in temperature. Seed recovered from dry soil, or kept without any medium, responded quickly at both temperatures. Light enhanced the germination of Phalaris minor seed. The seedbank subjected to rice (Oryza sativa) field management conditions lost vigour in comparison with the seed stored in laboratory. There was significant variability in seed viability when exposed to differential water management conditions in rice.     

Interaction of photoperiod and temperature in the control of growth and dormancy of Prunus species

Growth and dormancy induction of seedlings or micropropagated plants of three Prunus species were studied under controlled environment conditions. All the species tested, P. cerasus L. and P. insititia L. (two cultivars each), and P. avium L. were insensitive to photoperiod at high temperature and maintained continuous growth in both 10 and 24-h photoperiods at 21 °C. At lower temperatures, however, growth was controlled by the interaction of photoperiod and temperature, the species and cultivars varying somewhat in their responses. At 9 °C growth cessation was induced regardless of day-length conditions in the plum rootstocks ‘St. Julien A’ and ‘Weito’ as well as in the sour cherry rootstock ‘Weiroot’, whereas in the sour cherry rootstock ‘Gisela 5’ growth cessation took place in short day (SD) only. At intermediate temperatures (12 and 15 °C) growth cessation occurred in SD only in both sour cherry cultivars. In P. avium seedlings on the other hand, growth cessation in SD was only induced at 9 °C, continuous but reduced growth taking place also in SD at all higher temperatures. Growth rates increased progressively with increasing temperature under long day (LD) conditions in all species, and this was associated with increased internode length in LD compared with SD conditions. Production of new leaves was unaffected by photoperiod at high temperature, but was higher in LD than in SD at lower temperatures. After growth cessation at low temperature the plants developed winter buds and became dormant also in LD conditions. These results demonstrate that, like several species of the Pomoidae subfamily of the Rosaceae, these Prunus species are insensitive to short photoperiods at relatively high temperatures. However, the photoperiodic response of the Prunus species is highly temperature dependent, and the transition temperatures for shifts in the photoperiodic response mode vary among the species.     

Quantitative Detection of Fusarium Species in Wheat Using TaqMan

Fusarium head blight (FHB) of wheat and other small-grain cereals is a disease complex caused by several fungal species. To monitor and quantify the major species in the FHB complex during the growing season, real-time PCR was developed. TaqMan primers and probes were designed that showed high specificity for Fusarium avenaceum, F. culmorum, F. graminearum, F. poae and Microdochium nivale var. majus. Inclusion of an internal PCR control and serial dilutions of pure genomic DNAs allowed accurate determination of the concentration of fungal DNA for each of these species in leaves, ears as well as harvested grains of winter wheat. The DNA concentration of F. graminearum in grain samples correlated (r ²= 0.7917) with the incidence of this species on the grain as determined by isolation from individual kernels. Application of the TaqMan technology to field samples collected in 40 wheat crops in the Netherlands during the growing season of 2001 revealed that M. nivale var. majus predominated on leaves early in the season (GS 45-65). Ears and harvested grains from the same fields, however, showed F. graminearum as the major species. In 2002, grain samples from 40 Dutch fields showed a much wider range of species, whereas in ears from 29 wheat crops in France, F. graminearum was the predominant species. The concentration of DON correlated equally well with the incidence of the DON-producing species F. culmorum and F. graminearum in the grain samples (r ²= 0.8232) as well as with total DNA of both these species (r ²= 0.8259). The Fusarium TaqMan technology is an important tool to quantify and monitor the dynamics of individual species of the complex causing FHB in cereals during the growing season. This versatile tool has been applied in a comparison of different genotypes, but can also be applied to other disease management systems, e.g. fungicide treatments.     

The dynamics of N2O near the groundwater table and the transfer of N2O into the unsaturated zone: A case study from a sandy aquifer in Germany

The research area was the Fuhrberger Feld aquifer (FFA) in northern Germany. It is situated about 30km northeast of the city of Hannover and covers about 300km². Six multilevel sampling wells along a representative strip under predominantly arable land along a groundwater flow-line were sampled from the groundwater table down to a depth of 10m below the soil surface. We measured N₂O, CO₂, NO₃⁻, SO₄²⁻, DOC, pH, redox potentials and O₂ concentrations.N₂O accumulated at four out of six wells close to the groundwater table. About 20% of N₂O that occurred between the groundwater table and 7–8m below it resided in the top 0.4m. An exchange zone for N₂O at the interface between the saturated and the unsaturated zone extended 0.55 ± 0.22m below the groundwater table and acted as a source and sink for N₂O. N₂O below the exchange zone cannot be transferred from the groundwater to the atmosphere. The upward fluxes from the exchange zone into the unsaturated zone at the six wells ranged between 0.0009 and 0.3kg N₂O ha^{− 1} year^{− 1}. The yearly downward fluxes into the exchange zones had about the same order of magnitude as the upward fluxes. The upward and downward fluxes of N₂O at the (fluctuating) water table did cancel out each other, but this does not yet imply, that the N₂O fluxes at the soil surface also cancel out each other.N₂O–N:NO₃–N ratios were highly variable ranging from 0.0002 to 0.0417.A multiple regression for the monthly N₂O amounts in the exchange zone could explain 66% of the yearly variation. The significant variables were NO₃⁻, CO₂, pH, and O₂. Therefore, a combination of the land use (NO₃⁻), the geochemical boundary conditions (pH) and the type of denitrification reaction (O₂ and CO₂ indicate the importance of a heterotrophic denitrification process) governed the N₂O dynamics in the surface groundwater of the FFA and its transfer into the unsaturated zone.     

Evaluation of otoscope cone cleaning and disinfection procedures commonly used in veterinary medical practices: a pilot study

The objective of this study was to evaluate the relative efficacy of otoscope cone cleaning and disinfection methods commonly used in veterinary practices. Using sterile technique, 60 new gas-sterilized 4-mm otoscope cones were inoculated with a broth culture of 1.5 billion Pseudomonas aeruginosa bacteria per mL then allowed to dry for 10 min. Six study groups of 10 cones each were created. Group 1 served as positive control and received no cleaning or disinfection. Group 2 cones were wiped with sterile cotton-tipped applicators and gauze then rinsed with water. Group 3 cones were wiped with 70% isopropyl alcohol. Group 4 cones were scrubbed in a speculum cleaner with Cetylcide II solution (Cetylite Industries, Inc., Pennsauken, NJ). Groups 5 and 6 cones were soaked for 20 min in Cetylcide II and chlorhexidine gluconate 2% solutions, respectively. Using sterile technique and after 10-15 min drying time, the cones were swabbed in a consistent pattern, and samples were submitted for quantitative culture. Culture results showed no growth from cones soaked in Cetylcide II or chlorhexidine solutions. Two of the 10 cones wiped with alcohol, 3/10 cones wiped then rinsed with water, and 3/10 cones scrubbed with the speculum cleaner showed growth of P. aeruginosa. All (10/10) cones in the control group showed heavy growth of P. aeruginosa. These results show that P. aeruginosa can survive on otoscope cones cleaned and disinfected by several commonly used methods. Further study is needed to determine practical and optimal cleaning and disinfection methods for otoscope cones.     

Additional Indications for the Low Allergenic Properties of the Apple Cultivars Santana and Elise

Patients with Oral Allergy Syndrome (OAS) to fresh apple may tolerate low allergenic apple cultivars. We aimed to investigate if the low allergenic properties of Elise and Santana, as previously identified in a Dutch population, could be generalised within North West Europe within the birch pollen region with regard to both the prevalence and degree of sensitization. Prick-to-prick tests (PTP) were performed in eighty-five adult patients with OAS to fresh apple in Great Britain, Switzerland and Northern Italy, before the birch pollen season, using the putatively low allergenic apple cultivars Elise, Santana, Granny Smith, Modi and Mcintosh, as well as the putatively high allergenic apple cultivars Golden Delicious and Kanzi. No significant differences in percentages of negative responses of PTPs were found between the three countries. Negative responses did not differ from negative responses to the different apple cultivars we previously found in 2006/2007 in the Netherlands. The size of the PTPs of all apple cultivars tested were correlated to the size of the skin prick tests with birch pollen. These results add to the indications for the low allergenic properties of the low allergenic apple cultivars Santana and Elise, as the number of negative responses were reproducible in three countries within the birch pollen region and were similar to previous results in the Netherlands. These results justify oral challenge studies with Elise and Santana within the birch pollen region, to establish the low allergenic properties for the benefit for apple allergic consumers for definite conclusions.