**1. Introduction**

252 Current Frontiers in Cryopreservation

Zhang, Y. Z.; Zhang, S. C.; Liu, X. Z.; Xu, Y. Y.; Wang, C. L.; Sawant, M. S.; Li, J. & Chen, S.

practical methodology. Theriogenology 60(5):989–996.

L. (2003). Cryopreservation of flounder (Paralichthys olivaceus) sperm with a

Wels catfish (*Silurus glanis*) and pikeperch (*Sander lucioperca*) are two predator fish species cultivated in the traditional Central European pond aquaculture. Their role in the pond ecosystem is the control of the populations of smaller wild fish that enter ponds during their flooding in the Spring and would represent food competition for the cultured cyprinids. In addition, both species are highly priced for their excellent boneless meat, therefore, attempts are made to improve their culture and enhance yields (Horváth et al., 2002).

The process of induced propagation of wels catfish is based on a long-standing technology. However, certain problems can still appear: the method of collecting male gametes is still based on the removal of testes. By the application of this method a particular male can only be used once for propagation. Moreover, differentiation of sexes requires a great deal of experience and the danger of using an immature female with a less developed body structure is still present. As a result of listed problems the success of propagation becomes questionable.

In recent years successful experiments have been carried out in the pikeperch in many farms and research centers for the development of induced propagation in hatcheries. Synchronization of maturation of female individuals is not perfectly developed yet, thus, successful striping requires a constant attention and control. That is why minimizing the presence of males and securing sperm in a most simple way could focus attention on females.

Application of cryopreserved male gametes for fish propagation in hatcheries can serve as a solution for all mentioned difficulties and risks. History of fish sperm cryopreservation dates back to the beginning of 1950s, since then the sperm of more than 200 fish species has been cryopreserved successfully all over the world (Rana, 1995). In spite of this the application of cryopreserved fish sperm is still not very common in aquaculture in contract to e.g. the dairy cattle sector. Most studies done on cryopreservation of fish sperm put the emphasis on optimization of the process, on cryopreservation of a small amounts of male gametes, thus, rendering this technology to the level of laboratory experiments without any basic output for farmers.

Sperm Cryopreservation of Two European Predator Fish Species,

placed into liquid nitrogen.

counted after hatching.

amount of data.

**season in hatchery conditions** 

determined after hatching.

**2.1.4 Application in hatcheries** 

examined both in fresh and cryopreserved samples.

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 255

ratio of testis and body weight. After the squeezing of sperm the motility of gametes was defined as described above. After the collection of gametes the former described cryopreservation method was used with the addition that in case of samples collected in Tuka the effect of cooling time on motility and fertilizing capacity was also tested. Cooling time of samples varied between 3, 5 and 7 minutes. After the cooling period straws were

First propagation tests were completed in the Szajol farm of Fish-Coop Kft. Eggs were gained from fish by a routine propagation process. In the first experiments eggs were divided into 40, 80 and 120 g doses and each dose was propagated with 1 straw of cryopreserved sperm. Fertilized eggs were then incubated in 7 l Zug-jars. Hatching rate was

The second experimental procedure was performed in the Attala fish farm of Attala Hal Kft., when eggs were divided up to two 150 g doses and one of them was fertilized with one, while the other one with two straws of sperm. In both cases freezing time was 7 minutes. At

In the experimental procedure the cooling rate was measured, too. A straw was filled with cryopreservation medium. The K type sensor of a Digi-Sense DualLogR digital thermometer (Eutech Instruments, Singapore) was placed into the straw which was then laid onto a 3 cm high polystyrene frame floating on the surface of liquid nitrogen. The thermometer recorded temperature data with 1 second intervals. Temperature data were collected for 6 minutes since storing capacity of the memory of the thermometer allowed the recording of this

**2.1.3 Cryopreservation and analysis of sperm collected outside of the spawning** 

In 2006 wels catfish sperm was collected in January and March (aside from the spawning season) in the Köröm farm of Aqua-culture Kft. (Köröm Fish Farm, Local Government of Bőcs at present) from wels catfish kept in a flow-through intensive system. They were kept in tanks in a constant water temperature of 20 °C. The method applied for cryopreservation was the same as already mentioned with the difference that sperm was frozen for 7 minutes on the polystyrene frame before placing it into liquid nitrogen. Motility of sperm was

Propagation experiments were performed at the Szajol farm of Fish-Coop Kft. For this, cryopreserved samples originating from Szeged, 2005 and Köröm, 2006 were used. Eggs were collected from female fish by a routine propagation process. In the experimental procedure eggs were distributed into 250 and 350 g dosages and fertilized with one straw of sperm. Fertilized egg doses were then incubated in 7 l Zug-jars and hatching rates were

The aim of these experiments was to fertilize significant amounts of eggs (150-300 g) with large doses of cryopreserved sperm (5-ml straws) all over the country by joining to propagation work of a given farm. Reliability and repeatability of the method were also

hatching the ratio of hatching and deformed larvae was determined.

By the help of a successful cryopreservation method applicable in fish farms not only the reduction of propagation risks would become possible but also a long-term storage of gametes of substantial breeders as already applied in case of carp. Development of a sperm bank already employed at cattle breeding would also become feasible thus increasing the role and rate of rarely used selection methods of animal breeding in fisheries.

The objective of our work was the development of working protocols for the cryopreservation of wels catfish and pikeperch sperm that can be applied to aquaculture of these species. In both species, this development required a thorough knowledge of culture conditions of the given species, studies of the cryoresistance of sperm and adaptation of cryopreservation methods to hatchery practices.
