**6. Current status of fish sperm cryopreservation in Malaysia –the fish semen cryobank**

The current development of fish sperm cryopreservation in Malaysia is mainly focused on the creation of a fish semen cryobank. Such a cryobank for fish semen plays many important roles, especially for conservation of fish stocks and improve aquaculture. The establishment of a fish semen cryobank requires procurement of equipment and facilities for cryopreservation, skill development of the technical operators, choice of species for semen cryobanking, identification of the source for sample collection, developing of suitable cryopreservation procedure for the targeted species, proper record keeping and also proper maintenance of the semen cryobank.

### **6.1 Fish semen cryopresevation for conservation**

Cryopreservation technology provides long term *ex situ* conservation of indigenous and endangered species (Mongkonpunya et al., 2000; Tiersch et al., 2000). Cryopreserved semen could be stored indefinitely without deterioration provided proper maintenance and handling measures are well taken care of all time (Stoss & Donaldson, 1983; Armitage, 1987; Suquet et al*.,* 2000). In Malaysia, fish sperm cryopreservation was employed to save the genetics of the endangered and threatened indigenous species such as *P. jullieni* and *Tor*  spp*..* Fish species for gene banking and research are chosen on the basis of their threatened status and potential for aquaculture. Species with vulnerable, critically endangered (CR) and endangered (EN) status are prioritized in a conservation programme (Table 6). Species with protandrous behaviour throughout their life cycle such as the *Tenualosa toli* (Terubok) is also protected (MACD, 1996; Blaber et al., 2001; Wong, 2001). Other candidates of indigenous fish species which should be prioritized for semen cryopreservation are the Bala Shark (*Balantiocheilos melanopterus*)*,* the indigenous catfish (*Clarias macrocephalus*) and several *Betta*  spp*.* (*B. chini, B. hipposideros, B. livida, B. tomi* and *B. persephone*).

Semen samples could be stored for years via cryobanking before being used, and thus cryopreserved sperm banks can serve as insurance policies against unforseen catastrophes. However, very strict standardized protocols should be developed so that results are not biased by experimental and treatment variability but only by the inherent variability of the species. Further, these practices should be complemented with habitat conservation procedures (Bart, 2002). As it takes a long time to restore degraded ecosystems, the preserved semen is stored while upgrading of the ecosystems is going on. Both *in situ* and *ex situ* conservation should be applied complimentarily for sustainable management of the indigenous fisheries resources (Harvey, 1998). For an instance in the conservation programme of *P. jullieni,* introduction of closing season (from February to April each year) and conservation zones (12.4 km) at the identified spawning ground of *P. jullieni* at Pahang River have been implemented as the *in situ* conservation approach for the species (Zulkafli et al., 2010).

Conservation of indigenous freshwater fishes is a priority of the Department of Fisheries, Malaysia. Resulting from the success in breeding both *P. jullieni* and *Tor* spp. using cryopreserved semen, cryobanking of the semen of these species was initiated in 2008. However, those samples were collected from the cultured stock. Therefore, effort to further enrich the sperm cryobank through collection from wild stocks for different varieties and

**6. Current status of fish sperm cryopreservation in Malaysia –the fish semen** 

The current development of fish sperm cryopreservation in Malaysia is mainly focused on the creation of a fish semen cryobank. Such a cryobank for fish semen plays many important roles, especially for conservation of fish stocks and improve aquaculture. The establishment of a fish semen cryobank requires procurement of equipment and facilities for cryopreservation, skill development of the technical operators, choice of species for semen cryobanking, identification of the source for sample collection, developing of suitable cryopreservation procedure for the targeted species, proper record keeping and also proper

Cryopreservation technology provides long term *ex situ* conservation of indigenous and endangered species (Mongkonpunya et al., 2000; Tiersch et al., 2000). Cryopreserved semen could be stored indefinitely without deterioration provided proper maintenance and handling measures are well taken care of all time (Stoss & Donaldson, 1983; Armitage, 1987; Suquet et al*.,* 2000). In Malaysia, fish sperm cryopreservation was employed to save the genetics of the endangered and threatened indigenous species such as *P. jullieni* and *Tor*  spp*..* Fish species for gene banking and research are chosen on the basis of their threatened status and potential for aquaculture. Species with vulnerable, critically endangered (CR) and endangered (EN) status are prioritized in a conservation programme (Table 6). Species with protandrous behaviour throughout their life cycle such as the *Tenualosa toli* (Terubok) is also protected (MACD, 1996; Blaber et al., 2001; Wong, 2001). Other candidates of indigenous fish species which should be prioritized for semen cryopreservation are the Bala Shark (*Balantiocheilos melanopterus*)*,* the indigenous catfish (*Clarias macrocephalus*) and several *Betta* 

Semen samples could be stored for years via cryobanking before being used, and thus cryopreserved sperm banks can serve as insurance policies against unforseen catastrophes. However, very strict standardized protocols should be developed so that results are not biased by experimental and treatment variability but only by the inherent variability of the species. Further, these practices should be complemented with habitat conservation procedures (Bart, 2002). As it takes a long time to restore degraded ecosystems, the preserved semen is stored while upgrading of the ecosystems is going on. Both *in situ* and *ex situ* conservation should be applied complimentarily for sustainable management of the indigenous fisheries resources (Harvey, 1998). For an instance in the conservation programme of *P. jullieni,* introduction of closing season (from February to April each year) and conservation zones (12.4 km) at the identified spawning ground of *P. jullieni* at Pahang River have been implemented as the *in situ* conservation approach for the species (Zulkafli

Conservation of indigenous freshwater fishes is a priority of the Department of Fisheries, Malaysia. Resulting from the success in breeding both *P. jullieni* and *Tor* spp. using cryopreserved semen, cryobanking of the semen of these species was initiated in 2008. However, those samples were collected from the cultured stock. Therefore, effort to further enrich the sperm cryobank through collection from wild stocks for different varieties and

**cryobank** 

et al., 2010).

maintenance of the semen cryobank.

**6.1 Fish semen cryopresevation for conservation** 

spp*.* (*B. chini, B. hipposideros, B. livida, B. tomi* and *B. persephone*).

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


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

Table 6. List of indigenous freshwater fish species in Malaysia with critically endangered (CR), endangered (EN), vulnerable (VU) or data deficient (DD) status.


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

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 285

and topping up of liquid nitrogen in the storage dewars on periodically basis. Obtaining adequate funding and financial support to maintain the semen cryobank is part of the challenges to the institutions owned a semen cryobank. The monitoring of the viability of cryopreserved semen samples is also required from time to time to ensure the viability of stored samples is maintained. Post-thaw sperm motility was assessed for each batch of semen samples a week after cryo-storage and prior to be used for egg fertilization. The viability of the cryopreserved sperm was also evaluated from time to time through egg fertilization tests. A database that is able to provide good records of storage and withdrawals of samples from the semen cryobank is also a long term challenge in genebanking via cryopreservation (Kincaid, 2000). Proper record is essential for ease of

There are technical limitations to use cryopreserved semen in fish breeding as it requires involvement of skilled personnels. Therefore, training of operators or technicians in the related discipline is seen required in the technology extension programme. The small volume sample in straws (0.25 mL and 0.5 mL) is sufficient to be used in egg fertilization in laboratory based experiments and in genetic improvement programme of the targeted species. However, it is less practical to use cryopreserved semen for mass production of fry in aquaculture. Adaptation of the current developed protocols for practical application is thus important. The use of bigger straws volume or cryovials (5 mL or 10 mL) should be

Difficulties in getting wild stocks are the main constraints in fish semen cryobanking of the indigenous species with threatened or endangered status and those species with high market demand. These limitations have caused difficulties in obtaining the effective population size (Ne), which is very crucial in future restoration efforts for the species. High quality seed is essential to support aquaculture. For many species especially of those riverine species, which their induced breeding method is still not established, their source of seed supply is still depend on the wild caught stocks. In Malaysia, it is also difficult to obtain fry and broodstock from hatchery because the lack of well organised hatchery operation. Each hatchery tends to maintain their own breeders, which is always limited in numbers. As the consequences, this resulted in high inbreeding rates among the hatchery

In Malaysia, it is now increasingly difficult for fish breeders to locate and collect genetic materials from healthy or relatively undisturbed populations in the wild. The loss of genetic material in fish species can hinder the development of the aquaculture industries, especially fish farms and hatcheries. Many hatcheries often rely on too few breeders to reproduce, resulting in lower production, susceptibility to diseases and poor survival rates in the wild. As wild fish stocks disappear, it becomes even more difficult for hatcheries to find new breeders. At the same time breeding within small populations with limited genetic diversity results in inbreeding depression, i.e. genetic drift, producing small or stunted fish stocks. Therefore, fish genetic resources must be conserved and utilized sustainably because they are the key to maintaining the viability of cultured and natural fish populations. They

**7.4 Difficulties in obtaining semen samples from wild populations** 

samples retrieval in future.

**7.3 Technical limitations** 

considered instead.

stocks.

#### **6.2 Fish semen cryopreservation for aquaculture**

Fish gene banks offer vast potential benefits to hatcheries (Munkittrick & Moccia, 1984; Chao & Liao, 2001). It offers genetic variability to fish hatcheries around the world. The use of frozen semen in breeding programmes offers a means to further broaden the genetic base of the targeted species. Genetic improvement of broodstock or hatchery species for traits such as disease resistant, fast growth rate, salinity tolerance etc. could also make feasible with the establishment of the cryogenic sperm bank. The applications of sperm cryopreservation in aquaculture were also highlighted by Mongkonpunya et al. (2000). In the case of some species, males and females reach maturity over different periods of time, the cryopreserved semen could facilitate artificial fertilization and seed production (Tiersch, 2000). Besides, cryopreserved semen is easier to transport than live fish for culturing. This eliminates the stress to fish. The risk of transmitting diseases is also reduced by using cryopreserved semen.

On the other hand, the use of cryopreserved sperm also provides flexibility in breeding programmes, especially in producing hybrids with favourable characteristics for culturing such as higher viability, intensive growth rate, adaptive flexibility, early sexual maturation etc. (FAO, 1971). Hybridization in fish culture becomes feasible and more manageable with the utilization of cryopreserved semen. For example, in a breeding programme of *H. wetmorei*  carried out in our laboratory, *H. wetmorei* was cross-bred with the Javanese barb, *B. gonionotus*  successfully via the use of cryopreserved sperm and surrogate egg from *B. gonionotus*. Such breeding procedure was needed because mature female of *H. wetmorei* was not available during the induced breeding programme of the species. Therefore, it is no doubt that the use of cryopreserved sperm provides greater control in breeding programmes.

#### **7. Challenges to the gene banking of fish gamete via cryopreservation**

#### **7.1 Procedure optimization**

The principle and process behind semen cryopreservation sound rather simple, i.e. the storage of semen samples in ultra-low temperatures and liquid nitrogen (-196°C) is normally used. In avian species (such as chicken, fowl, turkey, goose and duck) and mammal livestock species (such as cattle, horse, boar, sheep and goat), protocols for their semen cryopreservation are well developed and established (Hammerstedt & Graham, 1992; Curry, 2000; Donoghue & Wishart, 2000; Woelders et al., 2003). In fish however, there are no standard protocols that are applicable to all species. Unlike terrestrial animals, it is more difficult to standardize the semen cryopreservation protocols in fish (Tiersch, 2000). This is because different fish species exhibited different responses to the same extenders and cryoprotectants. For an example, the protocol or diluents formulation which served optimal to *P. jullieni* may not be necessarily suitable and served optimum in other species. As such, developing the species-specific and reproducible sperm cryopreservation procedure is thus required for this purpose. These include the choice of the type of extender solution and cryoprotectant, the rates of freezing and thawing etc. In general, the optimization of cryopreservation protocol for a species involved a series of complex interactions among various factors in each step.

#### **7.2 Maintenance and proper recording**

It is less costly to maintain a semen cryobank in a long run compared to *in situ* conservation approach such as live genebank. However, some costs need to be allocated for replenishing and topping up of liquid nitrogen in the storage dewars on periodically basis. Obtaining adequate funding and financial support to maintain the semen cryobank is part of the challenges to the institutions owned a semen cryobank. The monitoring of the viability of cryopreserved semen samples is also required from time to time to ensure the viability of stored samples is maintained. Post-thaw sperm motility was assessed for each batch of semen samples a week after cryo-storage and prior to be used for egg fertilization. The viability of the cryopreserved sperm was also evaluated from time to time through egg fertilization tests. A database that is able to provide good records of storage and withdrawals of samples from the semen cryobank is also a long term challenge in genebanking via cryopreservation (Kincaid, 2000). Proper record is essential for ease of samples retrieval in future.
