**4. Discussion on semen cryopreservation of Malaysian freshwater fish species**

The successful semen cryopresevation of several Malaysian fish species e.g. *P. jullieni, T. tambroides, T. deuronensis*, *H. bleekeri*, *P. nasutus* and *H. wetmorei* using various methods of cryopresevation discussed above may be evaluated via analyses on the sperm motility, fertilization, hatching ability, etc.

#### **4.1 Quality of semen**

Sperm motility for the freshly collected semen from the healthy broodfish is usually ranged between 90 - 100% motility provided that the sperm sample was not activated prior to the actual cryopreservation processes or contaminated by water or urine while sampling was carried out. However from our observation, sperm motility of most freshwater fishes dropped to <10% after 24 h if the sample was not extended using suitable extender solution, even though the sample was kept cool in a refrigerator (0- 5°C).

Sperm concentration is one of the important characteristic that determines the sperm quality of an individual male (Billard, 1986; Suquet et al., 1992; Billard et al., 1995). The sperm concentrations of all Malaysian fish species studied were between 2.2 x 108 to 6.2 x 1010 sperm cells per mL and this is in agreement with the studies by Leung & Jamieson (1991). On the other hand, short lifespan after activation is the typical characteristic of spermatozoa of freshwater fish species. The duration of sperm motility of most freshwater fishes is normally <1 min after the sperms are activated (Billard & Cosson, 1992; Lahnsteiner & Patzner, 2008). Duration of sperm motility of *Tor* spp. was about 40-50 s and it slowed down drastically after 20 s of progressive movements (Chew et al., 2010b). For Isok barb, it was about 20 s and slowed down after 10 s of progressive movements. Table 3 shows the range of sperm concentrations and motility duration in each Malaysian species studied at FRI Glami Lemi.


A cryopreservation protocol developed for a species is considered success if the semen that cryopreserved according to the certain protocol could successfully fertilize eggs and produced offspring. Fresh semen is usually included in the control treatment. The optimal sperm to egg ratio used should be determined prior to fertilization. The sperm: egg ratio at approximately 250,000:1 is usually sufficient and worked well in most species in our laboratory. However, other sperm to egg ratios from 1000:1 to 500000: 1 were tested by Butts et al. (2009) and 100 000: 1 was found to yield the best fertilization performance in Atlantic cod. The dry fertilization method is favorable in the fertilization test for freshwater fish and thus was applied for all Malaysian fish species studied in our laboratory. In the dry fertilization method, both eggs and sperms were mixed well before hatchery water was later added into the sperm/egg mixture to water-harden the fertilized eggs. After rinsing with hatchery water, fertilized eggs were then incubated in aquarium (with or without using a hatching trough depended on species). Water temperature was kept at room temperature between 23 - 28°C throughout the period of incubation. The duration of embryo development varied between species. Therefore, the duration required for the fertilized egg

**4. Discussion on semen cryopreservation of Malaysian freshwater fish** 

The successful semen cryopresevation of several Malaysian fish species e.g. *P. jullieni, T. tambroides, T. deuronensis*, *H. bleekeri*, *P. nasutus* and *H. wetmorei* using various methods of cryopresevation discussed above may be evaluated via analyses on the sperm motility,

Sperm motility for the freshly collected semen from the healthy broodfish is usually ranged between 90 - 100% motility provided that the sperm sample was not activated prior to the actual cryopreservation processes or contaminated by water or urine while sampling was carried out. However from our observation, sperm motility of most freshwater fishes dropped to <10% after 24 h if the sample was not extended using suitable extender solution,

Sperm concentration is one of the important characteristic that determines the sperm quality of an individual male (Billard, 1986; Suquet et al., 1992; Billard et al., 1995). The sperm concentrations of all Malaysian fish species studied were between 2.2 x 108 to 6.2 x 1010 sperm cells per mL and this is in agreement with the studies by Leung & Jamieson (1991). On the other hand, short lifespan after activation is the typical characteristic of spermatozoa of freshwater fish species. The duration of sperm motility of most freshwater fishes is normally <1 min after the sperms are activated (Billard & Cosson, 1992; Lahnsteiner & Patzner, 2008). Duration of sperm motility of *Tor* spp. was about 40-50 s and it slowed down drastically after 20 s of progressive movements (Chew et al., 2010b). For Isok barb, it was about 20 s and slowed down after 10 s of progressive movements. Table 3 shows the range of sperm concentrations and motility duration in each Malaysian species studied at FRI

even though the sample was kept cool in a refrigerator (0- 5°C).

**3.6 Egg fertilization** 

**species** 

to hatch is also varied among different species.

fertilization, hatching ability, etc.

**4.1 Quality of semen** 

Glami Lemi.


\*Duration of progressive movement before slowed down and finally stopped

Table 3. Sperm concentration and motility duration of each freshwater fish species studied at FRI Glami Lemi.

Osmolality is another critical variable in sperm quality (Honeyfield & Krise, 2000). As seen in many studies, seminal plasma osmolality among males fish is highly variable (Aas et al., 1991). According to Babiak et al. (2001), the extender solution that worked well to cryopreserve spermatozoa of a species should posses the ability to maintain the sperm cell viability by inhibiting sperm motility. The key of success is via the use of an extender solution which is almost isotonic or mimicking the seminal plasma of that particular species. Therefore, it is crucial to know the seminal fluid osmolality of a species before media and diluents for that particular species can be formulated.

In all species of Malaysian fishes studied, sperm motility generally reduced after freezing and thawing process compared to the sperm motility before any freezing procedure. In *P. jullieni*, the motility of cryopreserved semen has reduced by approximately 45% compared to the fresh semen. A reduction of sperm motility by an average of 15% and 30% in *T. tambroides* and *T. deuronensis* respectively was observed. In general, sperm motility reduced between 10 - 70% on average in the species studied (Table 4)*.* These observations are similar to studies in several other species such as *Cyprinus carpio* L. (Wamecke & Pluta, 2003), *Oncorhynchus mykiss*  (Lahnsteiner et al., 1996) and *Scophthalmus maximus* (Dreanno et al., 1997).


Table 4. Sperm motility (%) of the freshwater fish species studied before freezing and after thawing procedures

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 281

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

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

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

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

important to produce viable cryopreserved sperm.

diet) are the key factors that produced healthy brood stocks.

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

significantly higher by using dry method compared to wet method.

knowledge could later help production of fry using cryopreserved semen.

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

**5.2 Good quality eggs** 

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 the storage period.

### **4.2 Fertilization ability and hatching rate**

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


Table 5. Fertilization and hatching performance using cryopreserved sperm in several freshwater fish species carried out at FRI Glami Lemi

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.
