Investigation of long-term stress induced by several stressors by determination of the concentration of different blood plasma components in a model of Prussian carp (Carassius auratus gibelio BLOCH, 1783) and Common carp (Cyprinus carpio L., 1758).

Several compounds (carbohydrates, proteins, hormones, etc.) were used in fish to quantify the level of stress. Our investigations focused on two parameters of the blood plasma: plasma glucose and serum/plasma fructosamine (SeFa) that has not been tested on fish as yet. Experiments were conducted on two fish species. The concentrations of these components were investigated on Common carp (Cyprinus carpio L., 1758) and on Prussian carp (Carassius auratus gibelio BLOCH, 1783) from the Gödöllö-Isaszeg pond system by creating conditions different from ideal. Stress effects caused a fluctuating tendency in blood plasma glucose levels each week for both Common carp and Prussian carp, thus, there was no steady growth. However, SeFa concentrations exactly followed stress effects, moreover, it tolerated short-term negative effects (handling of fish, blood sampling) and did not cause alterations at individuals blood samplings. This experimental method can offer assistance to farmers in the daily routine (e.g. in fish transport) and in the technology of propagation.

Production of fish chimeras from embryonic cells.

The efficiency of two cell-transplantation methods were compared for the production of embryonic cell derived fish chimeras. The classic microinjection technique (blastula stage donor cells are microinjected into blastula embryos) was compared to a novel aggregation method for fish developed by our group. This method utilises the ability of dechorionated fish embryos to aggregate. Morula cells dissociated in Ca2+, Mg2+ free medium were aggregated to recipient embryos of different developmental stage. Donor cells and recipient embryos of different developmental stages were used for most efficient incorporation in the chimeras. THe fate of donor cells derived from blastulae, was followed by labelling them with FITC-dextrane (FD). The most efficient transplantations were gained by using 16-32 cell stage recipients for aggregation (18% survival of chimeras at swim up stage). Labelled donor cells were contributing to the embryos in varying ratio. A comparison of the efficiency of aggregation was made between diploid-diploid, diploid-haploid and diploid-interspecfic (diploid) hybrid chimeras. In all three cases chimeras containing different proportion of donor cells were gained. After one day incubation the embryos were dissociated by trypsin digestion and number of labelled and non-labelled cells were counted under fluorescent microscope. Experiments were performed on Rosy barb (Barbus conchonius), Carp (Cyprinus carpio) and African catfish (Clarias gariepinus).

Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs.

Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses. Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses. Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs. Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E. coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter. Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo. Spatial expression of lacZ was assayed by histochemical staining. A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

Introducing foreign genes into fish eggs with electroporated sperm as a carrier.

A new method has been developed for introduction of foreign genes into fish eggs. The procedure is based on the incubation of fish sperm cells suspended in dilute citrate solution with plasmid DNA, followed by application of high-field-strength electrical pulses (electroporation) to increase DNA binding., uptake, or both. Tissue homogenates and genomic DNA extracts of free swimming fry developed from eggs fertilized with treated sperm was tested to evaluate the efficiency of gene transfer. Dot blot hybridization and gene expression assay demonstrated the presence and expression of the reporter genes introduced in 2.6 to 4.2% of several hundreds of tested larvae of common carp (Cyprinus carpio L.), African catfish (Clarias gariepinus), and tilapia (Oreochromis niloticus). No transgene has been found in the fry resulting from parallel experiments without sperm electroporation. This is the first report on successful application of electroporated sperm cells for production of transgenic fish.