**2.1.1 Methods of collection and cryopreservation of male gametes**

Gametes applied in the experiments were invariably retrieved directly from the testis removed from the abdomen after the decapitation of male wels catfish (not stripped). After removing it from the abdomen the testis was cut up and squeezed out through a dry gauze into a Petri-dish. After extraction of sperm motility of gametes was examined through a light microscope at 200× magnification.

Ten per cent methanol was used as a cryoprotectant and 6% fructose as a diluent. pH of the cryopreservation medium was adjusted to 7.73 by the help of 1 M NaHCO3 solution. From the diluted sperm treated this way 4 ml was pipetted into a 5-ml straw.

In the process of cryopreservation liquid nitrogen was poured into a polystyrene box on the top of which a polystyrene frame was placed with a height of 3 cm and the straws were laid on it. Samples were stored in a canister storage dewar until use.

Straws were thawed in 40 °C of water for 40 seconds. After thawing the closed ends of straws were cut up and their content was poured into a test-tube or directly onto the eggs used for propagation. Motility of thawed sperm was examined according to the method already described. The method mentioned here was compiled on the basis of former experiments of the cryopreservation group of the Department of Aquaculture on African catfish (Urbányi et al., 1999; Horváth & Urbányi, 2000).

#### **2.1.2 Experiments on cooling time and sperm-egg ratio**

Male gametes applied for cryopreservation in 2005 were collected from the farm in Tuka of Szarvas-Fish Kft. and the farm in Szeged of Szegedfish Kft. by joining to their propagation processes. Males were injected with 4 mg/body weight kg carp pituitary in one dose by the assistants of the farm before the extraction of sperm. Length and weight of the body and weight of the testis were measured. Gonadosomatic indices (GSI) were determined from the

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

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

Experiments on the cryopreservation of sperm of both species were carried out during the years 2005-2008 at various locations in Hungary. Details of experiments including their

Gametes applied in the experiments were invariably retrieved directly from the testis removed from the abdomen after the decapitation of male wels catfish (not stripped). After removing it from the abdomen the testis was cut up and squeezed out through a dry gauze into a Petri-dish. After extraction of sperm motility of gametes was examined through a

Ten per cent methanol was used as a cryoprotectant and 6% fructose as a diluent. pH of the cryopreservation medium was adjusted to 7.73 by the help of 1 M NaHCO3 solution. From

In the process of cryopreservation liquid nitrogen was poured into a polystyrene box on the top of which a polystyrene frame was placed with a height of 3 cm and the straws were laid

Straws were thawed in 40 °C of water for 40 seconds. After thawing the closed ends of straws were cut up and their content was poured into a test-tube or directly onto the eggs used for propagation. Motility of thawed sperm was examined according to the method already described. The method mentioned here was compiled on the basis of former experiments of the cryopreservation group of the Department of Aquaculture on African

Male gametes applied for cryopreservation in 2005 were collected from the farm in Tuka of Szarvas-Fish Kft. and the farm in Szeged of Szegedfish Kft. by joining to their propagation processes. Males were injected with 4 mg/body weight kg carp pituitary in one dose by the assistants of the farm before the extraction of sperm. Length and weight of the body and weight of the testis were measured. Gonadosomatic indices (GSI) were determined from the

dates and locations are provided in the descriptions of experiments on each species.

**2.1.1 Methods of collection and cryopreservation of male gametes** 

the diluted sperm treated this way 4 ml was pipetted into a 5-ml straw.

on it. Samples were stored in a canister storage dewar until use.

catfish (Urbányi et al., 1999; Horváth & Urbányi, 2000).

**2.1.2 Experiments on cooling time and sperm-egg ratio** 

role and rate of rarely used selection methods of animal breeding in fisheries.

cryopreservation methods to hatchery practices.

**2.1 Experiments on wels catfish sperm** 

light microscope at 200× magnification.

**2. Materials and methods** 

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 placed into liquid nitrogen.

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 counted after hatching.

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 hatching the ratio of hatching and deformed larvae was determined.

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 amount of data.

#### **2.1.3 Cryopreservation and analysis of sperm collected outside of the spawning season in hatchery conditions**

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 examined both in fresh and cryopreserved samples.

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 determined after hatching.

### **2.1.4 Application in hatcheries**

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

Sperm Cryopreservation of Two European Predator Fish Species,

water flow-through in the troughs was 0.25 l/minute.

routine practice of the farm.

survival rate (%) was measured.

experiment lasted for 4 days.

**2.1.6 Experiments on larval survival** 

**2.2 Experiments on pikeperch sperm** 

**2.2.1 Origin of fish** 

**Examination of non-feeding larval stage** 

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 257

with chopped tubifex in every 3 hours. To prevent infections 36% formalin treatment was applied in a concentration of 10 ml/trough in every 4 hours. In each trough velocity of water flow-through was 3 l/minute. Rearing in the trough lasted for 10 days according to the

Laboratory experiments were performed in the Department of Aquaculture. Troughs of the recirculation system applied in the experiment were 40 cm long, 15 cm deep and 10 cm wide (though water depth was 10 cm due to the outlet/stub). Due to photophobia of wels catfish larvae the system was located in a dark room. In the experiment 5×100 individuals (larvae hatched after the application of control and cryopreserved sperm) took part in the treatments. In the first 4 days fish were fed once in every three hours. At the morning and evening feeding they were fed with plankton while at other feeding times experimental fish were fed with Perla Proactiv 4.0 fish diet. Following this 4-day period fish were fed with the above mentioned diet (sometimes also with plankton or Artemia) 4 times a day. Velocity of

In addition to body length and weight condition factor, specific growth rate (S.G.R.) and

In this developmental stage no farm study was done due to the fact that the trough system was not suitable for the accommodation of such small larvae. Moreover, the hatchery

Larvae examined at non-feeding life stage were produced in June 2008 in the hatchery of TEHAG Kft. The method of propagation and utilization of cryopreserved sperm was the same as described at the examination of feeding larval stage. After hatching larvae were transported to the laboratory of the Department of Aquaculture in Gödöllő where the survival of fish was tested using the rearing system built in the previous year. The

Main effects of cooling time and the quantity of eggs on hatching rate was examined in the Szajol experiment (Part 2.1.2) by the help of two-way analysis of variance (P<0.05). In case of testis collected off season at two sampling times as described in Chapter 2.1.3 GSI values were compared by the help of a two-sample t-test. Hatching results gained from fertilization experiments with cryopreserved and control sperm described in Chapter 2.1.4 were analysed with a t-test, too. These statistical tests were performed using the software GraphPad Prism 4.0 for Windows. In the examinations of larval rearing results of survival were compared with Chi2-test (Kruskal Wallis test), while body length, body weight, condition factors and SGR

Experiments on pikeperch were conducted two times and in two places: the first place was the Keszthely-Tanyakereszt fish laboratory of Georgikon Faculty of Agriculture at Pannon

protocol for fry rearing does not use the method of rearing in troughs at this age.

values with t-tests (Chapter 2.1.5) using the software SPSS 13 for Windows.

University and the second one was the Attala hatchery of Attala Hal Kft.

examined. In each case one dose of eggs was fertilized with the content of one straw. In the frames of a routine propagation work fertilization with cryopreserved sperm was propagated in five different farms:


In the Attala experiment in 15 May 2007 cryopreserved sperm collected ourside of the spawning season was used for the fertilization of 200 g egg doses.

The experiment in Köröm was carried out in 17 May 2007 in which cryopreserved sperm was applied for fertilization also collected off season. Egg doses of 200 g were fertilized both for treated and for control groups.

Following that, egg doses of 200 g were fertilized in the Százhalombatta farm of TEHAG Kft. in 22 May 2007. This time cryopreserved sperm from Szeged was used in the experiments collected in 2006 in the spawning season.

In the fourth experiment in 21 May 2007 egg doses of 200-350 g were fertilized in the Ördöngös farm of Aranykárász Co with cryopreserved sperm collected off season.

The last experiment was performed in the hatchery of Szegedfish Kft. in 23 May 2007. In the research work egg doses of 150 g were fertilized with cryopreserved sperm deriving from Szeged.

In all cases our team personally joined to the propagation work of the farm, thus, we ensured that an adequate amount of cryopreserved sperm was used for the egg doses. Process of propagation was always performed according to the practice of the certain farm. One dose of eggs was fertilized with one straw of thawed sperm. In all experiments hatching percentage of larvae was determined and in the experiment in Attala fertilization rate at 4-8 cell stage was also examined.

### **2.1.5 Experiments on larval survival**

For the analysis of larval survival, growth and survival of feeding larvae in 2007 and nonfeeding wels catfish larvae in 2008 were examined.

#### **Analysis of feeding larval stage**

When examining feeding larval stage in farm conditions, larvae were produced in Százhalombatta farm of TEHAG Kft. by applying local propagation methods. During fertilization process 1 straw of sperm was added to 200 g of eggs. After fertilization the 200 g doses of eggs were placed into 7 l Zug-jars. For fertilization of individuals in the control group native sperm from males of the farm was used. On the second day after fertilization hatched larvae were counted then after hatching the non-feeding larvae were placed into larva-tanks. On the third day after hatching when larvae started their exogenous feeding the ones devoted for the experiment were counted and placed into troughs. 1000 individuals were placed into a 100 l trough with flow-through water in 3 replcates. The stock was fed with chopped tubifex in every 3 hours. To prevent infections 36% formalin treatment was applied in a concentration of 10 ml/trough in every 4 hours. In each trough velocity of water flow-through was 3 l/minute. Rearing in the trough lasted for 10 days according to the routine practice of the farm.

Laboratory experiments were performed in the Department of Aquaculture. Troughs of the recirculation system applied in the experiment were 40 cm long, 15 cm deep and 10 cm wide (though water depth was 10 cm due to the outlet/stub). Due to photophobia of wels catfish larvae the system was located in a dark room. In the experiment 5×100 individuals (larvae hatched after the application of control and cryopreserved sperm) took part in the treatments. In the first 4 days fish were fed once in every three hours. At the morning and evening feeding they were fed with plankton while at other feeding times experimental fish were fed with Perla Proactiv 4.0 fish diet. Following this 4-day period fish were fed with the above mentioned diet (sometimes also with plankton or Artemia) 4 times a day. Velocity of water flow-through in the troughs was 0.25 l/minute.

In addition to body length and weight condition factor, specific growth rate (S.G.R.) and survival rate (%) was measured.

#### **Examination of non-feeding larval stage**

256 Current Frontiers in Cryopreservation

examined. In each case one dose of eggs was fertilized with the content of one straw. In the frames of a routine propagation work fertilization with cryopreserved sperm was

In the Attala experiment in 15 May 2007 cryopreserved sperm collected ourside of the

The experiment in Köröm was carried out in 17 May 2007 in which cryopreserved sperm was applied for fertilization also collected off season. Egg doses of 200 g were fertilized both

Following that, egg doses of 200 g were fertilized in the Százhalombatta farm of TEHAG Kft. in 22 May 2007. This time cryopreserved sperm from Szeged was used in the experiments

In the fourth experiment in 21 May 2007 egg doses of 200-350 g were fertilized in the

The last experiment was performed in the hatchery of Szegedfish Kft. in 23 May 2007. In the research work egg doses of 150 g were fertilized with cryopreserved sperm deriving from

In all cases our team personally joined to the propagation work of the farm, thus, we ensured that an adequate amount of cryopreserved sperm was used for the egg doses. Process of propagation was always performed according to the practice of the certain farm. One dose of eggs was fertilized with one straw of thawed sperm. In all experiments hatching percentage of larvae was determined and in the experiment in Attala fertilization

For the analysis of larval survival, growth and survival of feeding larvae in 2007 and non-

When examining feeding larval stage in farm conditions, larvae were produced in Százhalombatta farm of TEHAG Kft. by applying local propagation methods. During fertilization process 1 straw of sperm was added to 200 g of eggs. After fertilization the 200 g doses of eggs were placed into 7 l Zug-jars. For fertilization of individuals in the control group native sperm from males of the farm was used. On the second day after fertilization hatched larvae were counted then after hatching the non-feeding larvae were placed into larva-tanks. On the third day after hatching when larvae started their exogenous feeding the ones devoted for the experiment were counted and placed into troughs. 1000 individuals were placed into a 100 l trough with flow-through water in 3 replcates. The stock was fed

Ördöngös farm of Aranykárász Co with cryopreserved sperm collected off season.

in Attala pond farm of Attala Fish Production and Trading Kft.

spawning season was used for the fertilization of 200 g egg doses.

in Köröm fish farm of the Local Government of Bőcs

propagated in five different farms:

for treated and for control groups.

Szeged.

collected in 2006 in the spawning season.

rate at 4-8 cell stage was also examined.

**2.1.5 Experiments on larval survival** 

**Analysis of feeding larval stage** 

feeding wels catfish larvae in 2008 were examined.

 in Százhalombatta farm of TEHAG Kft. in Ördöngös farm of Aranykárász Co. in Szeged farm of Szegedfish Kft.

> In this developmental stage no farm study was done due to the fact that the trough system was not suitable for the accommodation of such small larvae. Moreover, the hatchery protocol for fry rearing does not use the method of rearing in troughs at this age.

> Larvae examined at non-feeding life stage were produced in June 2008 in the hatchery of TEHAG Kft. The method of propagation and utilization of cryopreserved sperm was the same as described at the examination of feeding larval stage. After hatching larvae were transported to the laboratory of the Department of Aquaculture in Gödöllő where the survival of fish was tested using the rearing system built in the previous year. The experiment lasted for 4 days.

#### **2.1.6 Experiments on larval survival**

Main effects of cooling time and the quantity of eggs on hatching rate was examined in the Szajol experiment (Part 2.1.2) by the help of two-way analysis of variance (P<0.05). In case of testis collected off season at two sampling times as described in Chapter 2.1.3 GSI values were compared by the help of a two-sample t-test. Hatching results gained from fertilization experiments with cryopreserved and control sperm described in Chapter 2.1.4 were analysed with a t-test, too. These statistical tests were performed using the software GraphPad Prism 4.0 for Windows. In the examinations of larval rearing results of survival were compared with Chi2-test (Kruskal Wallis test), while body length, body weight, condition factors and SGR values with t-tests (Chapter 2.1.5) using the software SPSS 13 for Windows.

#### **2.2 Experiments on pikeperch sperm**

#### **2.2.1 Origin of fish**

Experiments on pikeperch were conducted two times and in two places: the first place was the Keszthely-Tanyakereszt fish laboratory of Georgikon Faculty of Agriculture at Pannon University and the second one was the Attala hatchery of Attala Hal Kft.

Sperm Cryopreservation of Two European Predator Fish Species,

concentration was examined in a Bürker-chamber in a 1000× dilution.

canister. After thawing the motility of samples was also examined.

**experiment)** 

hatching.

**3. Results** 

**2.2.5 Applied statistical methods** 

the help of a two-sample t-test (P0.05).

**3.1 Experiments on wels catfish sperm** 

motility of sperm from Szeged was 80%.

(ANOVA) (Chapter 2.2.2).

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 259

This research was done in April 2007 in the hatchery of Attala Hal Kft. Gametes used in the experiment originated from the same farm. Male and female fish were treated by the method applied in the farm in line with local hatchery propagation. In the previous experiment it was difficult to avoid mixing of sperm with urine so in this case stripping was performed by a silicon catheter (inside diameter: 1 mm, outside diameter: 1.5 mm) which was introduced into the sperm duct. Motility estimations were made by the method described earlier. Sperm

Sperm originating from 3 males were used in the experiment. It was diluted in a 1:1 ratio with the following composition of cryopreservation medium: 350 mM glucose, 30 mM Tris, pH 8.0 (titrated with ccHCl), methanol with a concentration of 10%. Diluted gametes were pipetted into 0.5-ml straws. Cryopreservation and thawing methods arranged to the method described in the previous chapter. Sperm was stored for 1 week in liquid nitrogen in a

Stripped eggs were divided into 10 and 30 g doses in 3 replicates and into a 50 g dose in one replicate. One dose of eggs was fertilized with one straw. Fresh sperm was used as a control. Each dose was poured into a 7 l Zug-jar for incubation. Hatching rate was counted after

Results of experiments were evaluated with Graphpad Prism 4.0 for Windows program. Effect of cryoprotectants and diluents on motility and fertilization and the effect of diluent ratio and cryoprotectants on hatching ratio was examined by a two-way analysis of variance

Results gained from the second experiment (Chapter 2.2.3), namely motility (cryopreserved and fresh sperm) and hatching results (in case of 10 and 30 g egg doses) were analysed by

The GSI (gonadosomatic index) of male individuals from Tuka was 2 ± 4%. The motility of sperm before cryopreservation was 90%, while after thawing this rate was 0% in case of 3 minutes, 40% in case of 5 minutes and 70% in case of 7 minutes long freezing time. The

In the experiments carried out in Szajol the highest hatching rate (51 ± 1%) was observed at 7 minutes freezing time and 40 g of dose of eggs, although in the case of 5 and 7 minutes of freezing time a very similar hatching rate (between 40 ± 0% and 51 ± 1%) was observed (Figure 1.). Only the cooling time had a significant main effect (P < 0.0001) on the results,

Hatching rate of propagated eggs was 94% in the case of fertilization with a single straw in Attala, while fertilization with two straws resulted 77% hatching rate. The control results

considering that 3 minutes long cooling time gave lower hatching rate.

**2.2.4 Application of cryopreserved sperm to hatchery conditions (preliminary** 

### **2.2.2 First experiment**

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

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 1000× dilution.

The following diluents were prepared:


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, Hungary).

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 at fresh sperm.

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.

#### **2.2.3 Second experiment**

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 the same as the process applied in the first experiment.

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 hatching rate was defined.

#### **2.2.4 Application of cryopreserved sperm to hatchery conditions (preliminary experiment)**

This research was done in April 2007 in the hatchery of Attala Hal Kft. Gametes used in the experiment originated from the same farm. Male and female fish were treated by the method applied in the farm in line with local hatchery propagation. In the previous experiment it was difficult to avoid mixing of sperm with urine so in this case stripping was performed by a silicon catheter (inside diameter: 1 mm, outside diameter: 1.5 mm) which was introduced into the sperm duct. Motility estimations were made by the method described earlier. Sperm concentration was examined in a Bürker-chamber in a 1000× dilution.

Sperm originating from 3 males were used in the experiment. It was diluted in a 1:1 ratio with the following composition of cryopreservation medium: 350 mM glucose, 30 mM Tris, pH 8.0 (titrated with ccHCl), methanol with a concentration of 10%. Diluted gametes were pipetted into 0.5-ml straws. Cryopreservation and thawing methods arranged to the method described in the previous chapter. Sperm was stored for 1 week in liquid nitrogen in a canister. After thawing the motility of samples was also examined.

Stripped eggs were divided into 10 and 30 g doses in 3 replicates and into a 50 g dose in one replicate. One dose of eggs was fertilized with one straw. Fresh sperm was used as a control. Each dose was poured into a 7 l Zug-jar for incubation. Hatching rate was counted after hatching.

### **2.2.5 Applied statistical methods**

Results of experiments were evaluated with Graphpad Prism 4.0 for Windows program. Effect of cryoprotectants and diluents on motility and fertilization and the effect of diluent ratio and cryoprotectants on hatching ratio was examined by a two-way analysis of variance (ANOVA) (Chapter 2.2.2).

Results gained from the second experiment (Chapter 2.2.3), namely motility (cryopreserved and fresh sperm) and hatching results (in case of 10 and 30 g egg doses) were analysed by the help of a two-sample t-test (P0.05).
