Application of an enzyme-immunoassay (EIA) for rapid screening of 5 alpha-pregnane-3,20-dione (DHP) in blood plasma of the Asian elephant, Elephas maximus.

Populations of African (Loxodonta Africana) and Asian Elephants (Elephas maximus) in zoos and safari parks are at risk due to their low reproductive success. To extend the limited knowledge of their reproductive physiology, the development of easy and practical methods for the analysis of the relevant reproductive hormones is essential to support e.g. assisted reproduction. For the measurement of 5 alpha-pregnane-3,20-dione (DHP), the predominant ovarian gestagen in both species, an enzyme-immunoassay (EIA) based on commercial reagents was applied. Advantages of this EIA are the small volume of plasma needed for evaluation (5 microliters) and the possibility of direct processing without an extraction stage. The lower limit of detection was 0.16 ng/ml, the mean recovery was 101% and the mean coefficients of variation were 7.3% (intra-assay) and 9.9% (inter-assay). In Asian elephants, DHP levels reached 15 ng/ml during the luteal phase and up to 21 ng/ml during pregnancy. Estrous cycle lengths based on the lowest DHP concentrations varied from 12 to 20 weeks (mean 15.4 +/- 2.3). In two Asian elephant cows a calf was still-borne. Thereafter, the animals reassumed ovarian activity after approximately 8 and 13 weeks, respectively. In one animal estradiol implants for hormonal contraception caused a down regulation of the ovarian function as demonstrated by an irregular pattern of DHP secretion over a period of 48 weeks. We propose the direct DHP-EIA as a suitable method for reproductive monitoring in elephants, as it can be easily established in laboratories.     

Isolation and characterization of bone marrow-derived equine mesenchymal stem cells

OBJECTIVE: To isolate and characterize bone marrow-derived equine mesenchymal stem cells (MSCs) for possible future therapeutic applications in horses. SAMPLE POPULATION: Equine MSCs were isolated from bone marrow aspirates obtained from the sternum of 30 donor horses. PROCEDURES: Cells were cultured in medium (alpha-minimum essential medium) with a fetal calf serum content of 20%. Equine MSC features were analyzed to determine selfrenewing and differentiation capacity. For potential therapeutic applications, the migratory potential of equine MSCs was determined. An adenoviral vector was used to determine the transduction rate of equine MSCs. RESULTS: Equine MSCs can be culture-expanded. Equine MSCs undergo cryopreservation in liquid nitrogen without altering morphologic characteristics. Furthermore, equine MSCs maintain their ability to proliferate and differentiate after thawing. Immunocytochemically, the expression of the stem cell marker CD90 can be detected on equine MSCs. The multilineage differentiation potential of equine MSCs was revealed by their ability to undergo adipogenic, osteogenic, and chondrogenic differentiation. CONCLUSIONS AND CLINICAL RELEVANCE: Our data indicate that bone marrow-derived stromal cells of horses can be characterized as MSCs. Equine MSCs have a high transduction rate and migratory potential and adapt to scaffold material in culture. As an autologous cell population, equine MSCs can be regarded as a promising cell population for tissue engineering in lesions of the musculoskeletal system in horses.     

Relationship between somaclonal variation and type of culture in cucumber

Highly inbred B line of cucumber was used to compare the effect of four types of in vitro culture on somaclonal variation. The plants were regenerated from the following types of culture: twelve- and eighteen-month-old liquid culture of meristematic clumps (LMC₁₂₍₁₈₎), ten-month-old embryogenic cytokinin-dependent suspension (CDS), eighteen-month-old embryogenic cytokinin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (CDS 1.7), twelve-month-old embryogenic auxin-dependent suspension (ADS), thirty six-month-old embryogenic auxin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (ADS 1.7) and recurrent leaf callus regeneration (RLC) – repeated 5 times. The differences in the incidence of the following properties were observed: the ploidy of R₀ plants, the segregation of new morphological traits in R₁ and the germination ability of R₁ seeds. R₁ families with the segregation of new phenotypes were most numerous in CDS (62.5%) and LMC₁₈ (57.9%), next in CDS1.7 (35.7%), while the smallest number was found in LMC₁₂ (11.1%) and RLC (3.4%).Tetraploid and mixoploid plants occurred in ADS1.7 and ADS (100%) whereas CDS and RLC were observed to contain only tetraploids, respectively 33.3% and 55.2%. There were no changes of ploidy after LMC₁₂, LMC₁₈ and CDS1.7. Among new phenotypes there were such that have not been described so far in cucumber: ginkgolike leaf (gll), yellow-green chlorophyll mutants (y-gc), serrate margin of corolla in male and female flowers (smc). This revised version was published online in August 2006 with corrections to the Cover Date.     

Field evaluation of antisense RNA mediated inhibition of GBSS gene expression in potato

Summary Granule-bound starch synthase (GBSS) catalyses the synthesis of amylose in starch granules. Analysis of antisense RNA mediated inhibition of GBSS gene expression in large numbers of tubers from in vitro grown, greenhouse grown and field grown transgenic potato plants revealed stable and total inhibition of GBSS gene expression in one clone. In three other transgenic genotypes partial and unstable inhibition was found. In these genotypes both GBSS activity and amylose content were remarkably reduced compared with the non-transformed control genotype. No relationship was found between the level of inhibition of GBSS gene expression and yield and dry matter content.     

Long-term impact of fertilization on activity and composition of bacterial communities and metabolic guilds in agricultural soil

To explore long-term impact of organic and inorganic fertilizers on microbial communities, we targeted both the total bacterial community and the autotrophic ammonia oxidizing bacteria (AOB) in soil from six treatments at an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO₃)₂, (NH₄)₂SO₄, unfertilized and unfertilized without crops. All plots, except the bare fallows, were cropped with maize. Effects on activity were assessed by measuring the basal respiration and substrate induced respiration (SIR) rates, and the potential activity of the AOB. To determine the bacterial community composition, 16S rRNA genes were used to fingerprint total soil communities by terminal restriction fragment length polymorphism analysis and AOB communities by denaturing gradient gel electrophoresis. The fertilization regimes had clear effects on both activity and composition of the soil communities. Basal respiration and r, which was kinetically derived as the exponentially growing fraction of the SIR-response, correlated well with the soil organic C content (r=0.93 and 0.66, respectively). Soil pH ranged from 3.97 to 6.26 in the treatments and was found to be an important factor influencing all microbial activities. pH correlated negatively with the ratio between basal respiration and SIR (r=0.90), indicating a decreased efficiency of heterotrophic microorganisms to convert organic carbon into microbial biomass in the most acid soils with pH 3.97 and 4.68 ((NH₄)₂SO₄ and sewage sludge fertilized plots, respectively). The lowest SIR and ammonia oxidation rates were also found in these treatments. In addition, these treatments exhibited individually different community fingerprints, showing that pH affected the composition of AOB and total bacterial communities. The manure fertilized plots harbored the most diverse AOB community and the pattern was linked to a high potential ammonia oxidation activity. Thus, the AOB community composition appeared to be more strongly linked to the activity than the total bacterial communities were, likely explained by physiological differences in the populations present.     

Impact of lakeshore development on green frog abundance

Many amphibian species exhibit metapopulation spatial dynamics and temporally are faced with local population extinction and re-colonization. These natural population fluctuations can exhibit stochastic effects when human-caused alteration and fragmentation of habitats occur during sensitive life-cycle events. In this study, we explored the effects of shoreline development on adult green frogs Rana clamitans melanota on lakes (n=24) of northern Wisconsin. We estimated green frog abundance using both auditory and direct observation surveys. The immediate shoreline habitat was mapped and placed into a Geographical Information System (GIS) for analysis. Adult green frog populations were significantly lower on lakes with varying degrees of shoreline house and cottage development than lakes with little or no development. A negative linear relationship existed between shoreline development densities and the number of adult green frogs. However, house and cottage densities alone did not directly explain this reduction. Analysis of variance (ANOVA) identified that the amount of suitable habitat, not development density, significantly affected green frog abundance. Therefore, greater development densities significantly decrease breeding habitat quality, resulting in lower adult frog abundance. These and other findings suggest that lakeshore development regulations are not protecting sensitive amphibian species.     

Numerical taxonomic studies in the generaTriticum L. andPisum L.

Summary Up till now numerical taxonomy has been applied to cultivated plants only to a limited extent though this method seems to be especially suitable for handling the great diversity of these plants. Moreover the theory of numerical taxonomy has not been studied sufficiently.Therefore studies with cultivated plants were begun with the aim to investigate the fitness of different numerical taxonomic techniques for their classification. Materials used: 1) 24 species ofTriticum and additionally 24 species ofAegilops 2) 1225Triticum taxa from Iran (mostlyT. aestivum) 3) 312 taxa ofPisum (mostlyP. sativum).Using 10 coefficients and 7 clustering methods inTriticum andAegilops in most cases a clear separation of both genera resulted as well as a species arrangement withinTriticum according to the genomes.The main aim when dealing with the wheat from Iran was the investigation of the influence of different character sets. At this the coefficient ofGower and the unweighted pair-group method using arithmetic averages (UPGMA) were applied. The separation of the different species is satisfying clear while the representatives of the different varieties ofT. aestivum could not always be found in one cluster. This effect increased when adding further characters sets.InPisum the influence of 11 similarity coefficients to the dendrogram formation has been investigated. With two-state characters the simple matching coefficient and with mainly multistate characters the Canberra metric gave best results (using UPGMA and information measures SUMRAT and SAMRAT).The hitherto existing results show the complicatedness of the evaluation of numerical taxonomic methods in their application to cultivated plants. Therefore generalizations are not yet possible.

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Numerisch-taxonomische Studien in den GattungenTriticum L. undPisum L.