Cumulus Oophorus Expansion of Bovine Oocytes Cultured in vitro: A SEM and TEM Study

Bovine cumulus-oocyte complexes (COCs) were aspirated from antral follicles (3–6 mm in diameter). COCs with a compact multilayered cumulus investment were cultured for 9, 13 and 24 hours, respectively, and processed for scanning or transmission electron microscopy after different periods of culture including a 0 hour control group. In all 0 hour control COCs initial formation of cumulus cell projections, blebs and microvilli were observed. The start of cumulus-oocyte disconnection in cattle was observed after 9 hours of in vitro culture. These connections were almost completely discrupted after 13 hours, when a continuous production of extracellular matrix was observed. The full expansion of corona radiata cells was not observed even after 24 hours. Some cumulus oophorus cells were bound together with junctions of the zonula adherens type after 24 hours when extracellular matrix was found only in deeper layers. The full expansion of corona radiata cells was not the prerequisite for disconnection of cumulus-oocyte complex. Inhalt: Kumulus oophorus Expansion von in vitro kultivierten Rinderoozyten: Eine SEM und TEM Untersuchung Kumulus-Oozyten-Komplexe (COC) von Rindern wurden aus antralen Follikeln aspiriert (Durchmesser 3–6 mm). COC's mit kompakten mehrschichtigen Kumulus wurden für 9, 13 und 24 Stunden kultiviert und für die Scanner- oder Transmissionselektronenmikroskopie vorbereitet. Verschiedene Kulturzeiten incl. einer 0-Stundengruppe als Kontrolle wurden untersucht. In allen Kontroll COC's wurden beginnende Formationen yon Kumuluszellprojektionen, Bläschen und Mikrovilli beobachtet. Nach 9-stündiger in vitro Kultur begann die Lösung zwischen Kumulus und Oozyte. Die Verbindungen waren nach 13 Stunden vollständig unterbrochen, wobei eine kontinuierliche Produktion extrazellulärer Matrix beobachtet wurde. Eine vollständige Expansion der Corona radiata Zellen wurde auch nicht nach 24 Stunden beobachtet. Nach 24 Stunden waren einige Cumulus Oophorus Zellen über Verbindungen des Zonula adherens Typs verknüpft, wobei extrazelluläre Matrix nur in den tieferen Zellschichten gefunden wurde. Eine vollständige Kumulusexpansion ist keine Voraussetzung für die Auflösung des Kumulus-Oozyten-Komplexes.     

Fertilizing capacity of the ejaculate of nutria (Myocastor coypus) after the removal of the seminal vesicles as evaluated by the penetration test and natural mating

The fertility of male coypu sperm following seminal vesicle extirpation was investigated using the penetration test into the egg of Syrian golden hamster (Mesocricetus auratus). Ejaculates were obtained from five males by means of electro-ejaculation under halothane narcosis. The results of the zona-free hamster eggs (ZFHE) penetration test showed that the ejaculates of all the surgically treated coypu males were fertile and that ZFHE value fluctuated from 54 to 76.6%. The results obtained in experiments with natural mating revealed that the extirpation of male coypu seminal vesicles did not affect their fertility. In total 47 foetuses were found post mortem in ten coypu females covered by surgically treated males, which on average represented 4.7 foetuses per female.     

Dynamics of chlorinated pesticide residues in long-term storage of canned food

Meat tins denoted as pork in natural juice, beef with bacon, luncheon meat and liver paté, all produced in a food-processing plant, were stored under definite conditions (temperature 21 degrees C, relative humidity 73%) for three years. Before the long-term storage and then in half-year intervals, the contents of the residues of chlorinated pesticides, including HCB (hexachlorobenzene), gamma-HCH (gamma isomer of hexachlorocyclohexane) and pp'DDE (1,1-dichloro-2,2-bis/p-chlorofenyl/-ethylene), were determined in the above-mentioned final products by the method of gas chromatography. As suggested by the records of the changes in the pesticide residue contents in the meat tins during their long-term storage, the average contents of all chlorinated carbohydrates gradually decrease with the length of storage and the "reduction" of residues varies with the kind of final product and with the pesticides. In HCB the reduction ranged from 18.4 to 34.4%, in gamma-HCH from 36.2 to 80.4% and in pp'DDE from 23.9 to 48.9%.     

‘Plantibodies’: a flexible approach to design resistance against pathogens

Engineering resistance against various diseases and pests is hampered by the lack of suitable genes. To overcome this problem we started a research program aimed at obtaining resistance by transfecting plants with genes encoding monoclonal antibodies against pathogen specific proteins. The idea is that monoclonal antibodies will inhibit the biological activity of molecules that are essential for the pathogenesis. Potato cyst nematodes are chosen as a model and it is thought that monoclonal antibodies are able to block the function of the saliva proteins of this parasite. These proteins are, among others, responsible for the induction of multinucleate transfer cells upon which the nematode feeds. It is well documented that the ability of antibodies to bind molecules is sufficient to inactivate the function of an antigen and in view of the potential of animals to synthesize antibodies to almost any molecular structure, this strategy should be feasible for a wide range of diseases and pests.Antibodies have several desirable features with regard to protein engineering. The antibody (IgG) is a Y-shaped molecule, in which the domains forming the tips of the arms bind to antigen and those forming the stem are responsible for triggering effector functions (Fc fragments) that eliminate the antigen from the animal. Domains carrying the antigen-binding loops (Fv and Fab fragments) can be used separately from the Fc fragments without loss of affinity. The antigen-binding domains can also be endowed with new properties by fusing them to toxins or enzymes. Antibody engineering is also facilitated by the Polymerase Chain Reaction (PCR). A systematic comparison of the nucleotide sequence of more than 100 antibodies revealed that not only the 3′-ends, but also the 5′-ends of the antibody genes are relatively conserved. We were able to design a small set of primers with restriction sites for forced cloning, which allowed the amplification of genes encoding antibodies specific for the saliva proteins ofGlobodera rostochiensis. Complete heavy and light chain genes as well as single chain Fv fragments (scFv), in which the variable parts of the light (V_L) and heavy chain (V_H) are linked by a peptide, will be transferred to potato plants. A major challenge will be to establish a correct expression of the antibody genes with regard to three dimensional folding, assembly and intracellular location.     

Histological studies on cow intestinal mucosa and electron microscopic studies of the ruminal epithelium of cows fed a deficient diet for a prolonged time]

Histological and histochemical studies were carried out on the gastro-intestinal mucosa of three experimental cows (roughages/maize silage) and three control animals. The animals were slaughtered on termination of the long-term trial. Mucosa samples were taken, for further study, from the rumen, the duodenum, the jejunum, the large intestine and the appendix. Histochemical analysis did not reveal any essential differences in the activities of non-specific esterase, alkaline and acid phosphatase and lactic acid dehydrogenase in the mucosa of the rumen, the large and the small intestine and the appendix of both the experimental animals and the controls. The experimental animals were found to exhibit a higher rate of glutamate-dehydrogenase activity in the ruminal mucosa and in the mucosa of the large and small intestine. A higher succinate dehydrogenase activity was observed in the ruminal mucosa of the experimental animals, relative to that of the controls, while the activity in the intestinal mucosa was decreased. Only slight changes were noted in the activity of the enzymatic systems tested. Electron microscopic studies did not reveal any differences in the ultrastructure of the epithelial cells of the ruminal mucosa in both the experimental animal and the controls.     

Testing of the protective effect of cell-free lyophilized vaccine against Marek's disease in field trials]

The protective effect of a lyophilized vaccine against Marek's disease ("Keramvac"--Pfizer), prepared from a cell-free turkey's herpesvirus (strain FC 126), was compared in field trials with losses in the group of non-vaccinated chickens. Under the indicated conditions, the vaccine had only a 50.86% effectiveness. The possible causes of the reduced vaccination effect are discussed with regard to the pathomorphological and virological findings suggesting, among others, an increased incidence of the symptoms of the classical form of Marek's disease in the population investigated.     

Biological features and life table parameters of the mealy plum aphidHyalopterus pruni on different apricot cultivars

Development, survival, reproduction rate, and population growth parameters of the mealy plum aphidHyalopterus pruni (Geoffroy) (Hom.: Aphididae) were evaluated on four different apricot cultivars (Tyrinte, Sakıt, Colomer, and Bebeco) under field conditions in the Van region of Turkey. Experiments were carried out on exterior leaves of trees, 1.5–2 m above the ground. Plexiglas clip-cells (25×6 mm) with the upper side covered by muslin were used in the experiments. The mealy plum aphid performed better on Tyrinte than on the other cultivars tested. The fastest development time (first instar to adult; 9.4 days), highest daily reproduction rate (2.6 offspring/aphid/day), and highest total fecundity (48.1 offspring/aphid) were obtained on Tyrinte. The intrinsic rate of increase — a good indicator of the growth potential of a population — of individuals fed on Tyrinte was significantly greater than that of individuals fed on cvs. Colomer and Bebeco. While mean generation times (T _o ) of populations on different cultivars were close to each other, the net reproductive rate was the highest (29.45 offspring/aphid/generation) on Tyrinte and the population doubling time on Tyrinte was 18.7%, 25.2% and 26.3% faster than those of individuals on other cultivars tested. The results obtained in this study indicated that Tyrinte appeared to be the most susceptible to the mealy plum aphid among the cultivars tested. http://www.phytoparasitica.org. posting Nov. 23, 2004.