**3.1 Experiments on wels catfish sperm**

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 motility of sperm from Szeged was 80%.

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

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

Sperm Cryopreservation of Two European Predator Fish Species,

from Szeged.

cryopreserved or fresh sperm.

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 261

hatching rate. However, according to the head of the farm the fertilization of control group was as bad as the result of the 20% hatching rate. On the basis of the results it was observed that sperm form Köröm (out of spawning season) had similar hatching rate to the sperm

Results of different hatching experiments depended on the propagation method and on the quality of sperm. Fertilization with cryopreserved sperm form Attala resulted 97 1% of fertilization rate while control fertilization rate was 93 1%. There was no significant difference between the two rates (P = 0.0084). In the same experiment the hatching rate of the larvae was 95 2% while in the control group this rate was 94 6%. There was no statistically significant difference between the hatching rate of larvae originating from

Fig. 2. The cooling profile used with 5-ml straws in the experiments on wels catfish sperm

also differ significantly (P = 0.0249) but now in favor of the control group.

The hatching rate of larvae originating from cryopreserved sperm in Köröm was 84 5%, while this rate in case of larvae originating from fresh sperm was 69 16%. There was no significant difference between the results. It was observed in the experiments carried out in Százhalombatta that hatching rate of larvae originating from cryopreserved sperm was 50 3%, while the result of the control group was 50 6%. There was also no statistically significant difference between the two groups. Hatching results of the experiments carried out in Ördöngös at the place of Aranykárász Kft. were about 57 22% in case of larvae originating from cryopreserved sperm and 22 18% in case of larvae originating from fresh sperm. In this experiment a significant difference was found (P = 0.05) in favor of the cryopreserved group. The hatching rate of larvae originating from cryopreserved sperm in Szeged was 75 3%, while this rate in case of the control group was 83 1%. These results

were 89% and 81%. It is worth to mention that the ratio of deformed larvae hatched from eggs fertilized with a single straw was only 2.4% (1.8% in control), while in the case of fertilization with two straws it was 11.2% (7.3% in control).

The cooling rate of a straw was approximately -23°C/minute (Figure 2.). It was observed that the temperature of the straw was only -45°C after 3 minutes while after 5 minutes it was -104°C.

Fig. 1. Hatch rates of wels catfish eggs fertilized with cryopreserved sperm. Each batch of eggs (40 or 80 g) was fertilized with one 5-ml straw of cryopreserved sperm. Columns indicate cooling times employed during cryopreservation (3, 5 or 7 minutes). Data are presented as Mean ± SD (N = 3).

When sperm was collected outside of the spawning season, the average weight of the testes of wels catfish catfish from Köröm was 20.4 g and the average weight of the fish was 2.52 kg, thus GSI rate of them was lower than 1% except one male. This low GSI rate have not had adverse effects on the quality of sperm. No significant difference was observed between GSI rate in January and in March (P = 0.4589). The motility of fresh sperm varied between 50% and 90%. Two of the sperm samples selected for cryopreservation were excluded from further examinations because the motility of these samples was the lowest (50 - 60%). This low rate was caused likely by injuring the cells during squeezing of the testes. Some of the cryopreserved samples were thawed 5 days after freezing and their motility was about 50%.

Sperm frozen in 2005, in Szeged and 2006, in Köröm were used for the propagation experiments. Hatching rate varied between 70 - 80%, except for one sample with 20% of

were 89% and 81%. It is worth to mention that the ratio of deformed larvae hatched from eggs fertilized with a single straw was only 2.4% (1.8% in control), while in the case of

The cooling rate of a straw was approximately -23°C/minute (Figure 2.). It was observed that the temperature of the straw was only -45°C after 3 minutes while after 5 minutes it was

Fig. 1. Hatch rates of wels catfish eggs fertilized with cryopreserved sperm. Each batch of eggs (40 or 80 g) was fertilized with one 5-ml straw of cryopreserved sperm. Columns indicate cooling times employed during cryopreservation (3, 5 or 7 minutes). Data are

When sperm was collected outside of the spawning season, the average weight of the testes of wels catfish catfish from Köröm was 20.4 g and the average weight of the fish was 2.52 kg, thus GSI rate of them was lower than 1% except one male. This low GSI rate have not had adverse effects on the quality of sperm. No significant difference was observed between GSI rate in January and in March (P = 0.4589). The motility of fresh sperm varied between 50% and 90%. Two of the sperm samples selected for cryopreservation were excluded from further examinations because the motility of these samples was the lowest (50 - 60%). This low rate was caused likely by injuring the cells during squeezing of the testes. Some of the cryopreserved samples were thawed 5 days after freezing and their motility was about 50%. Sperm frozen in 2005, in Szeged and 2006, in Köröm were used for the propagation experiments. Hatching rate varied between 70 - 80%, except for one sample with 20% of

fertilization with two straws it was 11.2% (7.3% in control).


presented as Mean ± SD (N = 3).

hatching rate. However, according to the head of the farm the fertilization of control group was as bad as the result of the 20% hatching rate. On the basis of the results it was observed that sperm form Köröm (out of spawning season) had similar hatching rate to the sperm from Szeged.

Results of different hatching experiments depended on the propagation method and on the quality of sperm. Fertilization with cryopreserved sperm form Attala resulted 97 1% of fertilization rate while control fertilization rate was 93 1%. There was no significant difference between the two rates (P = 0.0084). In the same experiment the hatching rate of the larvae was 95 2% while in the control group this rate was 94 6%. There was no statistically significant difference between the hatching rate of larvae originating from cryopreserved or fresh sperm.

Fig. 2. The cooling profile used with 5-ml straws in the experiments on wels catfish sperm

The hatching rate of larvae originating from cryopreserved sperm in Köröm was 84 5%, while this rate in case of larvae originating from fresh sperm was 69 16%. There was no significant difference between the results. It was observed in the experiments carried out in Százhalombatta that hatching rate of larvae originating from cryopreserved sperm was 50 3%, while the result of the control group was 50 6%. There was also no statistically significant difference between the two groups. Hatching results of the experiments carried out in Ördöngös at the place of Aranykárász Kft. were about 57 22% in case of larvae originating from cryopreserved sperm and 22 18% in case of larvae originating from fresh sperm. In this experiment a significant difference was found (P = 0.05) in favor of the cryopreserved group. The hatching rate of larvae originating from cryopreserved sperm in Szeged was 75 3%, while this rate in case of the control group was 83 1%. These results also differ significantly (P = 0.0249) but now in favor of the control group.

Sperm Cryopreservation of Two European Predator Fish Species,

between hatching rates (Figure 4.).

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 263

The volume of the sperm stripped from pikeperch males in the second experiment was very low (less than 1 ml/individual). The motility of fresh sperm was 45 30%. Similarly to the previous experiment the sperm was mixed with urine again. Motility of thawed pikeperch sperm was very low (0 – 2%) in the samples containin the cryoprotectant DMSO, while motility of sperm frozen in presence of methanol was 40%, independently from rate of dilution. The highest hatching rate (41 22%) was observed with the use of methanol and 1:1 dilution rate, although the statistical analysis has not shown significant differences

Fig. 4. Fertilization rates of pikeperch eggs fertilized with cryopreserved sperm in the second experiment. The cryoprotectants methanol and dimethyl-sulfoxide (DMSO) and dilution ratios of 1:1 and 1:9 were compared. Data are expressed as Mean ± SD (N = 3).

difference (P = 0.1135) between the motility of fresh and cryopreserved sperm.

In the hatchery experiment Stripping sperm with silicon catheter resulted that the motility was 63 ± 10%. Concentration of sperm was 1.8571 ± 0.1538 × 1010, while the number of eggs/g was 1367 ± 54, thus the number of sperm for an egg was 3.396 × 105 in the case of 10 g dose of eggs, 1.132 × 105 in the case of 30-g dose of eggs and 6.792 × 104 in the case of 50-g dose of eggs. Motility of sperm after thawing was 53 ± 5%, thus there was no significant

Fertilization of the dose of 10 g of eggs with a single straw resulted 47 4%, while in the case of the dose of 30 g eggs resulted 55 3% hatching rate (Figure 5.). There was no

In the experiments on larval survival, statistically significant difference (P = 0.034) was observed on feeding larvae in laboratory conditions regarding 10-day body length. The results showed that larvae originating from cryopreserved sperm had a longer body. During non feeding larval period final body length (P < 0.001) and final weight (P = 0.018) differed significantly in the two groups in favor of larvae originating from cryopreserved sperm. There was no difference between the larvae originating from cryopreserved or fresh sperm in terms of larval survival.

## **3.2 Experiments on pikeperch sperm**

In the first experiment, in spite of all efforts sperm was mixed with urine during stripping, thus the motility of pikeperch sperm was 50 ± 17%. The motility of the best thawed sample was 28 ± 21 %, which was cryopreserved with glucose diluent and DMSO as cryoprotectant, but statistically significant difference was not be observed among the treatmenrts.

The density (spermatozoa/ml) of pikeperch sperm samples counted in a Burker chamber were the following: 1st male: 0.9375 × 1010, 2nd male: 1.0100 × 1010, 3rd male: 0.7037 × 1010, 4th male: 0.6687 × 1010.

The highest fertilization rate (43 ± 12%) was observed also in the case of using a combination of glucose diluent and DMSO as cryoprotectant (Figure 3.). During statistical analysis of the data it was found that only the cryoprotectant had a significant effect (P = 0.0338) on the ratio of the fertilization.

Fig. 3. Fertilization percentages of pikeperch eggs fertilized with cryopreserved sperm in the first experiment. The cryoprotectants dimethyl-sulfoxide (DMSO) and methanol (MeOH) and glucose, KCl or sucrose extenders were compared. Data are expressed as Mean SD (N = 3).

In the experiments on larval survival, statistically significant difference (P = 0.034) was observed on feeding larvae in laboratory conditions regarding 10-day body length. The results showed that larvae originating from cryopreserved sperm had a longer body. During non feeding larval period final body length (P < 0.001) and final weight (P = 0.018) differed significantly in the two groups in favor of larvae originating from cryopreserved sperm. There was no difference between the larvae originating from cryopreserved or fresh sperm

In the first experiment, in spite of all efforts sperm was mixed with urine during stripping, thus the motility of pikeperch sperm was 50 ± 17%. The motility of the best thawed sample was 28 ± 21 %, which was cryopreserved with glucose diluent and DMSO as cryoprotectant,

The density (spermatozoa/ml) of pikeperch sperm samples counted in a Burker chamber were the following: 1st male: 0.9375 × 1010, 2nd male: 1.0100 × 1010, 3rd male: 0.7037 × 1010, 4th

The highest fertilization rate (43 ± 12%) was observed also in the case of using a combination of glucose diluent and DMSO as cryoprotectant (Figure 3.). During statistical analysis of the data it was found that only the cryoprotectant had a significant effect (P = 0.0338) on the

Fig. 3. Fertilization percentages of pikeperch eggs fertilized with cryopreserved sperm in the first experiment. The cryoprotectants dimethyl-sulfoxide (DMSO) and methanol (MeOH) and glucose, KCl or sucrose extenders were compared. Data are expressed as Mean SD

but statistically significant difference was not be observed among the treatmenrts.

in terms of larval survival.

male: 0.6687 × 1010.

ratio of the fertilization.

(N = 3).

**3.2 Experiments on pikeperch sperm** 

The volume of the sperm stripped from pikeperch males in the second experiment was very low (less than 1 ml/individual). The motility of fresh sperm was 45 30%. Similarly to the previous experiment the sperm was mixed with urine again. Motility of thawed pikeperch sperm was very low (0 – 2%) in the samples containin the cryoprotectant DMSO, while motility of sperm frozen in presence of methanol was 40%, independently from rate of dilution. The highest hatching rate (41 22%) was observed with the use of methanol and 1:1 dilution rate, although the statistical analysis has not shown significant differences between hatching rates (Figure 4.).

Fig. 4. Fertilization rates of pikeperch eggs fertilized with cryopreserved sperm in the second experiment. The cryoprotectants methanol and dimethyl-sulfoxide (DMSO) and dilution ratios of 1:1 and 1:9 were compared. Data are expressed as Mean ± SD (N = 3).

In the hatchery experiment Stripping sperm with silicon catheter resulted that the motility was 63 ± 10%. Concentration of sperm was 1.8571 ± 0.1538 × 1010, while the number of eggs/g was 1367 ± 54, thus the number of sperm for an egg was 3.396 × 105 in the case of 10 g dose of eggs, 1.132 × 105 in the case of 30-g dose of eggs and 6.792 × 104 in the case of 50-g dose of eggs. Motility of sperm after thawing was 53 ± 5%, thus there was no significant difference (P = 0.1135) between the motility of fresh and cryopreserved sperm.

Fertilization of the dose of 10 g of eggs with a single straw resulted 47 4%, while in the case of the dose of 30 g eggs resulted 55 3% hatching rate (Figure 5.). There was no

Sperm Cryopreservation of Two European Predator Fish Species,

was used during the experiments, is suitable for these 5-ml straws.

identification as males.

in the hatcheries.

incurred cryodamage.

wels catfish fertilization.

ones collected in the spawning time.

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 265

be extracted from surgically removed testes (Legendre et al., 1996). As it was mentioned in the Introduction, this can lead to several problems such as shortage of sperm during induced spawning to unnecessary killing of immature females due to their erroneous

Cryopreservation of the sperm of catfish species has been studied extensively. Several studies have been published on the cryopreservation of wels catfish sperm (Krasznai & Márián, 1985; Linhart et al., 1993; Ogier de Baulny et al., 1999; Linhart et al., 2005), however, they all reported the use of minute amounts of gametes and did not test practical utilization

It can be concluded according to the measured freezing parameters that a longer cooling time is needed for the safe cryopreservation of sperm in 5-ml straws because the temperature is not low enough (-45ºC ) after 3 minutes. The 7 minutes cooling time, which

Large amounts of eggs can be fertilised safely with a single 5-ml straw. It was observed in these experiments that the amount of the eggs, fertilised with one straw can be increased because the 2 ml sperm that can be found in a straw contained enough spermatozoa to fertilise 120 g eggs. The use of 5-ml straws has been tested on several fish species including the rainbow trout *Oncorhynchus mykiss* (Wheeler & Thorgaard, 1991; Lahnsteiner et al., 1997; Cabrita et al., 2001) or the paddlefish *Polyodon spathula* (Horváth et al., 2010), however, all previous works report a more or less reduced fertilizing capacity of sperm cryopreserved in these straws as compared to the conventional 0.5-ml French straws. The reaction of sperm to cryopreservation in different straw types seems to be species specific with the sperm of the wels catfish being especially resistant to the

In the experiments no significant decrease was experienced in the quality of sperm after thawing. One of the reasons of this is that proper cooling time was successfully defined, which resulted the best fertilization rate. Thus, it can be said that cryopreserved sperm does

According to these experiments wels catfish catfish sperm collected outside of spawning season is as suitable for cryopreservation and for fertilization at fish farms similarly to the

After the successful cryopreservation and thawing of large amounts of sperm the next step is to carry out safe fertilization with this sperm on large scale. In the experiments doses of eggs between 150 - 350 g were fertilised with a single straw. According to literature data 100 - 200 g eggs can safely be incubated in one 7-l Zug-jar (Szabó, 2000). Cryopreserved sperm showed similar hatching rates to control in every experiment. These results prove that maximum sperm-egg ratio was not reached that might cause a decrease in hatching rate. According to the experiments it can be said that the improved method is suitable for

After improving the freezing method of sperm the next task was to examine whether the growth and survival of larvae originated from cryopreserved sperm reaches that of larvae originated from fresh sperm. The research was extended to both the feeding and non-

not decrease the hatching rate compared to the traditional, routine method.

statistically significant difference between the results of the different doses however the result of t-test (P = 0.05701) was very close to the significance level. A hatching rate of 87% was observed in the case of fertilization the dose of 50 g eggs with one thawed straw although in this case there were no replicates in the experiment. It was observed, however, that egg batches of different weight behaved differently in the hatching jars. While egg batches of 10 g stuck together in spite of the attempted elimination of egg stickiness, those in batches of 30 g or 50 g freely rolled on each other, thus improving oxygen supply of fertilized eggs and developing embryos. Thus, it is recommended to use larger batches of eggs for fertilization with cryopreserved sperm, which in turn would facilitate the acceptance of this technology in the aquaculture practice.

Fig. 5. Hatch rates of pikeperch eggs fertilized with cryopreserved sperm in a commerical hatchery during routine spawning work. Egg batches of 10, 30 or 50 g were used for fertilization (N = 3).

#### **4. Discussion**

#### **4.1 Experiments on wels catfish sperm**

Hatchery propagation of catfish species including the wels catfish faces several problems. Males of catfish species are typically oligospermic and sperm cannot be stripped but has to

statistically significant difference between the results of the different doses however the result of t-test (P = 0.05701) was very close to the significance level. A hatching rate of 87% was observed in the case of fertilization the dose of 50 g eggs with one thawed straw although in this case there were no replicates in the experiment. It was observed, however, that egg batches of different weight behaved differently in the hatching jars. While egg batches of 10 g stuck together in spite of the attempted elimination of egg stickiness, those in batches of 30 g or 50 g freely rolled on each other, thus improving oxygen supply of fertilized eggs and developing embryos. Thus, it is recommended to use larger batches of eggs for fertilization with cryopreserved sperm, which in turn would facilitate the

Fig. 5. Hatch rates of pikeperch eggs fertilized with cryopreserved sperm in a commerical hatchery during routine spawning work. Egg batches of 10, 30 or 50 g were used for

Hatchery propagation of catfish species including the wels catfish faces several problems. Males of catfish species are typically oligospermic and sperm cannot be stripped but has to

fertilization (N = 3).

**4.1 Experiments on wels catfish sperm** 

**4. Discussion** 

acceptance of this technology in the aquaculture practice.

be extracted from surgically removed testes (Legendre et al., 1996). As it was mentioned in the Introduction, this can lead to several problems such as shortage of sperm during induced spawning to unnecessary killing of immature females due to their erroneous identification as males.

Cryopreservation of the sperm of catfish species has been studied extensively. Several studies have been published on the cryopreservation of wels catfish sperm (Krasznai & Márián, 1985; Linhart et al., 1993; Ogier de Baulny et al., 1999; Linhart et al., 2005), however, they all reported the use of minute amounts of gametes and did not test practical utilization in the hatcheries.

It can be concluded according to the measured freezing parameters that a longer cooling time is needed for the safe cryopreservation of sperm in 5-ml straws because the temperature is not low enough (-45ºC ) after 3 minutes. The 7 minutes cooling time, which was used during the experiments, is suitable for these 5-ml straws.

Large amounts of eggs can be fertilised safely with a single 5-ml straw. It was observed in these experiments that the amount of the eggs, fertilised with one straw can be increased because the 2 ml sperm that can be found in a straw contained enough spermatozoa to fertilise 120 g eggs. The use of 5-ml straws has been tested on several fish species including the rainbow trout *Oncorhynchus mykiss* (Wheeler & Thorgaard, 1991; Lahnsteiner et al., 1997; Cabrita et al., 2001) or the paddlefish *Polyodon spathula* (Horváth et al., 2010), however, all previous works report a more or less reduced fertilizing capacity of sperm cryopreserved in these straws as compared to the conventional 0.5-ml French straws. The reaction of sperm to cryopreservation in different straw types seems to be species specific with the sperm of the wels catfish being especially resistant to the incurred cryodamage.

In the experiments no significant decrease was experienced in the quality of sperm after thawing. One of the reasons of this is that proper cooling time was successfully defined, which resulted the best fertilization rate. Thus, it can be said that cryopreserved sperm does not decrease the hatching rate compared to the traditional, routine method.

According to these experiments wels catfish catfish sperm collected outside of spawning season is as suitable for cryopreservation and for fertilization at fish farms similarly to the ones collected in the spawning time.

After the successful cryopreservation and thawing of large amounts of sperm the next step is to carry out safe fertilization with this sperm on large scale. In the experiments doses of eggs between 150 - 350 g were fertilised with a single straw. According to literature data 100 - 200 g eggs can safely be incubated in one 7-l Zug-jar (Szabó, 2000). Cryopreserved sperm showed similar hatching rates to control in every experiment. These results prove that maximum sperm-egg ratio was not reached that might cause a decrease in hatching rate. According to the experiments it can be said that the improved method is suitable for wels catfish fertilization.

After improving the freezing method of sperm the next task was to examine whether the growth and survival of larvae originated from cryopreserved sperm reaches that of larvae originated from fresh sperm. The research was extended to both the feeding and non-

Sperm Cryopreservation of Two European Predator Fish Species,

the fish farms mentioned in the work for their support.

*Aquaculture*, Vol.201, pp. 301–314, ISSN: 0044-8486

*Aquaculture*, Vol.247, pp. 243-251, ISSN: 0044-8486

Blackwell, ISBN 0-85238-282-0, Oxford, UK

pp. 25-28, ISSN: 0133-1922

177-184, ISSN: 0011-2240

ISSN: 0990-7440

*Aquaculture Research*, Vol.31, pp. 317-324, ISSN: 1355-557X

*Aquatic Living Resources*, Vol.16, pp. 457-460, ISSN: 0990-7440

method was tested in hatchery conditions.

**6. Acknowledgment** 

**7. References** 

the Pikeperch (*Sander lucioperca*) and the Wels Catfish (*Silurus glanis*) 267

rate of larvae originating from cryopreserved sperm reaches and in some cases exceeds that of control larvae. This result proves the practical usage of the cryopreservation method.

Pikeperch sperm has successfully been cryopreserved for the first time, and the developed

This work was supported by the NKFP 4/006/2004, RET 12/05 and the Bolyai János Fellowship of the Hungarian Academy of Sciences. The authors express their gratitude to

Cabrita, E.; Robles, V.; Alvarez, R. & Herráez, M.P., (2001). Cryopreservation of rainbow

Horváth, Á. & Urbányi, B. (2000). The effect of cryoprotectants on the motility and fertilizing

Horváth, Á.; Miskolczi, E. & Urbányi, B. (2003). Cryopreservation of common carp sperm.

Horváth, Á.; Wayman, W.R.; Urbányi, B.; Ware, K.M.; Dean, J.C. & Tiersch, T.R. (2005). The

Horváth, L.; Tamás, G. & Seagrave, C. (2002). *Carp and pond fish culture, Second edition*,

Krasznai, Z. & Márián, T. (1985). Kísérletek a lesőharcsa (*Silurus glanis* L.) spermájának

Lahnsteiner, F.; Weismann, T. & Patzner, R.A., (1997). Methanol as cryoprotectant and the

Legendre, M.; Linhart, O. & Billard, R. (1996). Spawning and management of gametes,

Linhart, O.; Billard, R. & Proteau, J.P. (1993). Cryopreservation of European catfish (*Silurus glanis* L.) spermatozoa. *Aquaculture*, Vol.115, pp. 347-359, ISSN: 0044-8486 Linhart, O.; Rodina, M.; Flajshans, M.; Gela, D. & Kocour, M. (2005). Cryopreservation of

succes of embryos. *Cryobiology*, Vol.51, pp. 250-261, ISSN: 0011-2240 Ogier de Baulny, B.; Labbé, C. & Maisse, G. (1999). Membrane integrity, mitochondrial

fishes. *Aquaculture Research*, Vol.28, pp. 471-479, ISSN: 1355-557X

trout sperm in large volume straws: application to large scale fertilization.

capacity of cryopreserved African catfish *Clarias gariepinus* (Burchell 1822) sperm.

relationship of cryoprotectants methanol and dimethyl sulfoxide and hyperosmotic extenders on sperm Cryopreservation of two North-American sturgeon species.

mélyhűtéses tartósítására és az évszaktól független szaporításra. *Halászat*, Vol.31,

suitability of 1,2 ml and 5 ml straws for cryopreservation of semen from salmonid

fertilized eggs and embryos in Siluroidei, *Aquatic Living Resources*, Vol.9, pp. 59-80,

European catfish *Silurus glanis* sperm: Sperm motility, viability, and hatching

activity, atp content, and motility of the European catfish (*Silurus glanis*) testicular spermatozoa after freezing with different cryoprotectants, Cryobiology, Vol.39, pp.

feeding larval periods. The results in both cases were that there is no difference in the survival of larvae fertilised with cryopreserved or fresh sperm. In the non-feeding larval period the growth of larvae from cryopreserved sperm exceeded the growth of the control, and in feeding larvae body length was higher compared to the control results.

According to these experiments the survival rate of larvae originating from cryopreserved sperm is as high as in the control and growth level of them in some cases showed better results compared to the control.
