**7. References**

266 Current Frontiers in Cryopreservation

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,

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

During the improvement of the cryopreservation technique of pikeperch sperm in laboratory cryoprotectant DMSO showed better fertilization rates than methanol but fertilization experiments in hatcheries showed opposite results. Literature data can be found on successful usage of both cryoprotectants in several fish species. The objective of this thesis is the usage of cryopreserved pikeperch sperm in hatcheries and according to the results of the experiments in the whole it was concluded that methanol and 1:1 dilution rate

A significant variation was observed in motility after thawing and in hatching rate in the first experiments. This variability is caused by mixing of sperm with urine. This problem can successfully be eliminated when the stripping of sperm is conducted with a silicone catheter. According to this method the sperm is stripped with this silicone catheter directly from the testes preventing the mixing of sperm with urine or feces. One year later the use of catheter

It was observed that the increasing of the amount of eggs fertilised with a single 0..5-ml straw resulted in improved hatching rates. The reason for this can be that different amounts of eggs behaved differently in Zug-jars. The dose of 10 g of eggs were slightly stuck together, the dose of 30 g of eggs stuck in smaller batches while the dose of 50 g of eggs rolled freely. In spite of the fact that the 50 g of eggs sample had not replicates, these results

It is supposed that the eggs in the middle of the 10-g batches were more sensitive for oxygen

Another explanation for these results is that methanol in smaller eggs samples was in higher concentration, thus the toxic effects were more drastic than in larger samples. The lower sperm-egg ratio in larger egg samples had no influence on the results, suggesting that the

A method has been developed for the cryopreservation of wels catfish sperm that can be used in the practice of fish farms. It is possible to fertilize 150-300 g eggs with sperm cryopreserved in large volumes (5-ml straws). The motility and hatching rate of frozen sperm correspond with the currently used routine method of fertilization with fresh sperm. The survival and growth parameters of wels catfish larvae originating from cryopreserved sperm was investigated for the first time. According to this study it can be said that survival

suggest that fertilisation of larger amounts of eggs result in better hatching rates.

amount of sperm was in surplus in the case of smaller egg samples.

and in feeding larvae body length was higher compared to the control results.

results compared to the control.

**4.2 Experiments on pikeperch sperm** 

is suitable for freezing pikeperch sperm.

resulted in substantially better hatching rates.

deficiency than the more loose larger egg samples.

**5. Conclusion** 


**14**

*Negeri Sembilan,* 

*Malaysia* 

**Sperm Cryopreservation of Some Freshwater** 

It has been estimated that spermatozoa can last from 200-32,000 years (Stoss & Donaldson, 1983; Suquet et al*.,* 2000). According to Kopeika et al. (2007), several methods of fish sperm storage has been practised including stored in medium with saturated gases, preservation at temperature above zero, in a frozen state as well as by drying. However, the low temperature approaches have been successful in fish sperm cryopreservation. Thus, cryopreservation technology offers the best means for long term storage of fish semen.

To date, successful cryopreservation of fish semen were reported in more than 200 freshwater species and 40 marine species worldwide (Gwo, 2000). Even though in general many successes have been achieved in fish semen cryopreservation, the technique remains as a method that is difficult to be standardized and use in all types of fishes. This is due to the fact that cryopreservation of sperms from different fish species required different conditions, where the protocol needs to be established individually. Even the "general protocol" of cryopreservation of fish sperm summarized by Kopeika et al. (2007) encompassed many variations when different species of fish are involved, particularly in the

In view of need to develop individual protocol for successful cryopreservation of fish semen and considering Malaysia has a rich fish fauna with many of them unique to this tropical region, cryopreservation of fish gametes will require detailed study to create new protocol for each fish species intended for semen cryopreservation. To date in Malaysia semen cryopreservation has only been reported for several freshwater fish species, namely *Probarbus jullieni, Tor tambroides, T. deuronensis*, *Hemibagrus nemurus*, *Pangasius nasutus, Hypsibarbus wetmorei, Barbonymus gonionotus* and *Clarias gariepinus.* It has been demonstrated that semen cryoperservation plays an important role for the genetic conservation of these fish species.

Cryopreservation technology for fish semen is still not well explored in Malaysia and can be considered as new if compared to the domesticated terrestrial livestocks. Henceforth, this has opened up a new field to be explored with potential applications in aquaculture and in the conservation of the national fisheries genetic resources. Cryopreserved semen could facilitate artificial fertilization especially when mature male fishes are not available or unable to provide viable semen during certain periods of the breeding season. Semen cryopreservation may also be useful for fertilization to produce hybrids of various fish species. It also helps in reducing

use of medium ingredients for the cryopreservation.

**1. Introduction** 

**Fish Species in Malaysia** 

Poh Chiang Chew and Abd. Rashid Zulkafli

*Freshwater Fisheries Research Division, FRI Glami Lemi, Jelebu,* 

