**5.2 Good quality eggs**

280 Current Frontiers in Cryopreservation

For all Malaysian fish species studied, the post-thaw motility rates of the cryopreserved sperm demonstrated similar values (p>0.05) even after a year of cryostorage as long as the semen samples are submerged well in the liquid nitrogen and without disturbance during

In both Malaysian Mahseer and Isok barb, egg fertilization ability and hatching rates were found significantly lower (p<0.05) by using cryopreserved sperm compared with those fertilized using fresh sperm. The speed of embryos development was similar among the fertilized eggs using both cryopreserved and fresh sperm. Besides that, no significant difference (p>0.05) was found in the egg fertilization percentages between newly cryopreserved semen and semen samples after a year of cryo-storage in both species. The performance of egg fertilization and hatching by using cryopreserved semen in four species

Species Fertilization (%) Hatching (%) Javanese barb 12 – 100 5 – 75 African catfish 21 - 37 19 – 32 Isok barb 1.2 – 10 0.8 – 4.6 Malaysian Mahseer 20 – 55 20 – 53

Table 5. Fertilization and hatching performance using cryopreserved sperm in several

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

the fertilization trials and knowledge in culturing the targeted species.

cryopreserved separately in order to maintain its inherent variability.

Quantitatively the numbers of hatchings from the cryopreserved semen were low, but qualitatively the hatchlings are visually normal and physically active and healthy as those from the fresh semen. Fingerlings produced from the cryopreserved semen from all four species showed normal development compared with those produced from the fresh semen.

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

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

of freshwater fish species studied in our laboratory is shown in Table 5.

freshwater fish species carried out at FRI Glami Lemi

**5.1 Viability of the cryopreserved sperm** 

the storage period.

**4.2 Fertilization ability and hatching rate** 

Good quality egg is also essential for successful fertilization when cryopreserved semen is used to produce offsprings. Same as sperm quality, egg quality is also the best during the peak of spawning seasons of the particular species. Thus fertilization trial is best to perform during spawning seasons of the fish. This is especially true and applicable for the seasonal bred species. In *P. jullieni*, significant higher fertilization rate (80-95%) was observed during the peak of the spawning season compared to the initial (50-60%) and toward the end of breeding season (30-50%) of the species. Healthy broodfish produced good quality eggs. Good management practice in broodstocks maintenance and good diet (i.e. high protein diet) are the key factors that produced healthy brood stocks.

#### **5.3 Handling of sperm and egg during the fertilization trials**

Good control in the timing of egg stripping and thawing of cryopreserved semen is important while using cryopreserved semen to fertilize eggs. Both egg and sperm should be made available at the same time in order to produce high fertilization rates. Spermatozoa of freshwater fishes are usually activated by the hypotonicity of their surrounding media (Morisawa & Suzuki, 1980; Morisawa et al*.,* 1983a; Morisawa et al*.,* 1983b). Most of the time and in majority species, hatchery water is sufficient as the sperm activation media and used in the sperm-egg mixture during fertilization process with cryopreserved semen. However, the use of suitable medium other than hatchery water for sperm activation is sometimes critical in some species (Lahnsteiner et al., 2003). This is observed in *T. tambroides* where specially formulated medium produced significant higher fertilization rate compared to the use of hatchery water as the sperm activation medium (Chew et al., 2010b). The effects of several media on egg fertilization ability had also reported by Billard (1983) on rainbow trout. Fertilization technique used in the fertilization trials is also one of the contributing factors to the success. Our studies showed that fertilization and hatching ability were significantly higher by using dry method compared to wet method.

#### **5.4 Knowledge in culturing of the targeted species**

Knowledge on the reproductive biology, broodstock management and husbandry, larva rearing and nursery of the particular fish species to be studied is a prerequisite for successful fertilization using cryopreserved semen. The age of maturity, breeding season (for seasonal bred species), factors that trigger and promote gonad maturation such as the type of nutrition and water quality where the brood fish is maintained are important factors that guaranteed gonad maturation and good health of the broodfish. Besides, well established artificial spawning method of the targeted species is also essential and such knowledge could later help production of fry using cryopreserved semen.

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 283

populations in the country is the ultimate aim of the Department of Fisheries, Malaysia. At present, semen samples from 88 *Tor* spp., 43 *P. jullieni*, 8 *P. nasutus* and 14 *H. wetmorei* have been collected and cryopreserved in the sperm cryobank of FRI Glami Lemi (Table 7).

The use of cryopreserved semen could support conservation efforts through stock enhancement and repopulation in areas where the species have declined or disappeared. In the breeding and restocking programmes, attempts to save the wild populations have so far largely focused on captive breeding or spawning of wild broodstock and subsequent release of hatchery-reared offsprings into the wild. Hatchery production of fry will support stock enhancement. Consequently, this will hopefully eliminate the need to harvest seed stock

Species Status

EN VU VU EN CR VU CR CR CR CR VU EN EN EN DD CR VU VU

EN EN EN

from the wild and the translocation of non-indigenous species for such programme.

Table 6. List of indigenous freshwater fish species in Malaysia with critically endangered

Species Number of fish Volume *Tor* spp. 88 150 mL *P. jullieni* 43 350 mL *P. nasutus* 8 18 mL *H. wetmorei* 14 30 mL

Table 7. Current status of cryogenic fish sperm bank of FRI Glami Lemi, Malaysia (since 2008)

(CR), endangered (EN), vulnerable (VU) or data deficient (DD) status.

*Balantiocheilos melanopterus* (Bala shark) *Betta chini* (Chini mouthbrooder)

*Betta livida* (Emerald-sport fighting fish) *Betta persephone* (Black fighting fish)

*Encheloclarias curtisoma* (Soft fin walking catfish) *Encheloclarias kelioides* (Soft fin walking catfish)

*Parasphromerus harveyi* (Harvey's licorice gouramy)

*Sundoreonectes tiomanensis* (Tioman cave loach)

*Discherodontus halei* (Spot-fin barb)

*Encheloclarias prolatus* (Catfish)

*Scleropages formosus* (Asian Arowana)

(Source: DOF, Malaysia; Chong et al., 2010 )

*Probarbus jullieni* (Isok barb)

*Helicophagus waandersii Cyclocheilichthys enoplus Leptobarbus hoeveni* (Mad barb)

*Betta hipposideros* 

*Betta tomi* (Pikehead)

*Eleotris melanosoma* 

*Silurus furness Redigobius bikalanus Phallostethus dunckeri* 
