**3. Results**

258 Current Frontiers in Cryopreservation

The stock of pikeperch taking part in the first experiment originated from Aranyponty Kft. (Sáregres-Rétimajor) (8 females and 10 males: 1424-1870 g). Males were anesthetized by clove oil then sperm was manually stripped and collected with an automatic pipette. Care

Motility of fresh sperm was estimated after activating it with water. Motility was examined on slides in a 20× dilution at 200× magnification by the help of a Zeiss Laboval 4 microscope (Carl Zeiss, Jena, GDR). Density of sperm was examined by the Bürker-chamber method in a

Methanol and dimethyl-sulfoxide (DMSO) were used as cryoprotectants in a concentration of 10%. All chemicals used in the experiments were purchased from Reanal Zrt. (Budapest,

Sperm (200 μl) mixed with a cryopreservation medium (200 μl cryoprotectant, 1600 μl diluent) in a ratio of 1:9 (Horváth et al., 2003; Urbányi et al., 2006) was pipetted into individually marked 0.5-ml straws after 3 minutes of equilibration time. Samples were cryopreserved in a polystyrene box filled with liquid nitrogen. A 3 cm high polystyrene frame was placed onto the top of the nitrogen then straws were laid on this frame where temperature was around -165°C. The time of cryopreservation was 3 minutes. After the freezing process straws were placed into liquid nitrogen and stored there until being used. Thawing was carried out in a 40C water bath for 13 seconds (Horváth et al., 2003; Horváth et al., 2005). After thawing sperm motility was examined with the same method as described

Eggs were distributed to Petri-dishes with a diameter of 5 cm with 200-350 eggs/dose. Fertilization was made with thawed sperm of a half straw (250 μl) right after taking the straw out of the water. Sperm was poured on the egg doses then the gametes were activated with 1 ml of water. Next eggs were allowed to stick to the bottom of the Petri-dish by taking care of the eggs being located in one layer. Fertilization rate was counted at neurula stage.

The second experiment was performed in line with pikeperch propagation in a hatchery. In this research sperm of 4 males and eggs of 1 female were applied. In this case only glucose was used as a diluent with 10% concentration of methanol and DMSO cryoprotectants. Sperm was diluted in a 1:1 and 1:9 ratios. The process of cryopreservation and thawing was

Eggs were divided into 10 g doses (about 10 000 eggs according to my counts) and taken into plastic bowls. Each dose was fertilized by a thawed straw of samples (0.5 ml) then these doses were placed into 7 l Zug-jars until hatching. Finally hatched larvae were counted and

was taken not to contaminate the gametes with urine or feces.

Glucose diluent (350 mM glucose, 30 mM Tris, pH 8,0)

Sacharose diluent (300 mM sacharose, 30 mM Tris, pH 8,0)

KCl diluent (200 mM KCl, 30 mM Tris, pH 8,0)

**2.2.2 First experiment** 

1000× dilution.

Hungary).

at fresh sperm.

**2.2.3 Second experiment** 

hatching rate was defined.

the same as the process applied in the first experiment.

The following diluents were prepared:
