**5. Dependence of egg fertilization on semen cryopreservation methods**

For the sperm cryopreservation of the fish species such as *P. jullieni* and *Tor* spp., several factors will determine the successful of the egg fertilization. The contributing factors that brought to the success of fish hatching using cryopreserved semen include viability of the post-thaw cryopreserved semen, good quality eggs, handling of the sperm and egg during the fertilization trials and knowledge in culturing the targeted species.

#### **5.1 Viability of the cryopreserved sperm**

Semen collection for cryopreservation should be done during the peak of spawning seasons of the particular species because the quality and quantity of spermatozoa are the highest at that particular time. The cryopreserved sperm cells tend to deteriorate very quickly and loss their motilily within 10-30 min after thawing procedure. Therefore, in quantification of the quality of the post-thaw sperm, the post-thaw motility shall be evaluated soon after thawing procedure in order to obtain good results. Similarly in the fertilization trials using cryopreserved semen, the mixture of semen to the eggs should be performed as quickly as possible once the frozen semen is thawed. Semen samples from each male broodfish were cryopreserved separately in order to maintain its inherent variability.

Successful cryopreservation procedures can maintain high quality of cryopreserved semen and this is revealed in high post-thaw sperm motility percentages. Successful cryopreservation procedures balance between the formation of ice crystals within the cells against excessive dehydration, which damages cellular structures (Tiersch, 2000). Each step involved in the cryopreservation procedures that was discussed in section 3 is equally important to produce viable cryopreserved sperm.
