**2. Semen cryopresevation of freshwater fish species of Malaysia**

Malaysia has close to a hundred river systems, two natural lakes (Lake Chini and Lake Bera) and a vast freshwater water bodies and peat swamps (Baluyut, 1983). On fish biodiversity, Malaysia has approximately 616 reported freshwater fish species (Froese & Pauly, 2003). Most of the inland fisheries resources are dominated by the cyprinids and silurids (Khoo et al., 1987). Some endemic species are found in rivers, lakes and peat swamps. In the past decades intensive development activities such as deforestation and land clearing for oil palm plantation or other agricultural uses, indiscriminate logging within and around the catchment areas and construction of dams for hydroelectricity, has led to many riverine fish species suffer high risk of extinction (Khoo et al., 1987; Jackson & Marmulla, 2001). These development activities have resulted in habitat destruction, deterioration of water quality, water pollution and sedimentation, especially during rainy season when runoff is increased. All these certainly have direct great impacts on some fish species and the impacts are irreversible (Ho, 1995). Apart from the environmental degradation resulting from development, other causes of loss in inland fisheries biodiversity are attributed to overexploitation due to strong market demand, the use of illegal and destructive fishing gears such as poisoning and electro-shocker and the introduction of invasive exotic species (Dudgeon, 2002; Allan et al*.,* 2005). The indigenous species such as Isok barb (*Probarbus jullieni*), Malaysian Mahseer (*Tor* spp.), Mad barb (*Leptobarbus hoeveni*), Hampala barb (*Hampala macrolepidota*), knife fish (*Chitala lopis*), climbing perch (*Anabas testudineus*), snakeheads (*Channa* spp.), Asian arowana (*Scleropages formosus*), the pangasiid catfishes (*Pangasius nasutus, Pangasigodon waandersii*), giant river catfish (*Wallago leerii*), large headed walking catfish (*Clarias macrocephalus*) and giant gouramy (*Osphronemus goramy*) have dwindled in great numbers continuously due to unsustainable fishing activities. At present, most Malaysian fish species could only be conserved probably in the inaccessible or remote areas of the country.

It is no doubt that continuing habitat destruction, overfishing and competition for food from the invading fish species are leading to loss of inland fisheries biodiversity even before much of them could be documented. The lack of data on the indigenous species will subsequently impede efforts to better utilize and manage the nation's inland fisheries resources in a sustainable manner, and can eventually resulting in threatened, endangered or extinction of fish species in Malaysia. With respect to National Biodiversity Policy, it is therefore vital to protect and safeguard the indigenous fisheries resources while the species still exist in the wild. Realizing the risk of depleting fish stocks from natural waters, studies in domestication, management and husbandry of broodstocks, artificial breeding, grow out, nutrition and effort in stock enhancement via restocking of selected indigenous freshwater fish species have been carried out by the Department of Fisheries, Malaysia ever since 1980's. The species which have been studied and documented were the indigenous catfishes (*Clarias macrocephalus* and *C. batrachus*)*,* Tropical bagrid catfish (*Hemibagrus nemurus*)*,*  Hampala barb (*Hampala macrolepidota*), Isok barb (*Probarbus jullieni*)*,* Malaysian Mahseer (*Tor tambroides*)*,* local pangasiid catfish (*Pangasius nasutus*) (Pathmasothy & Omar, 1982; Pathmasothy, 1985; Saidin, 1986; Thalathiah et al., 1988; Mohamad-Zaini, 1992; Thalathiah et al., 1992; Ahmad-Ashhar, 1992; Ahmad-Ashhar & Haron, 1994; Ahmad-Ashhar, 1996; Suhairi, 1996; Suhairi et al., 1996; Ahmad-Ashhar, 1998).

In 2007, cryopreservation of semen was implemented as one of the *ex situ* conservation approach, with the exotic species such as the Javanese barb (*Barbonymus gonionotus*) and African catfish (*Clarias gariepinus*) used as the model species to examine the various suitable formulations diluents and skill development in semen cryopreservation. These two species were chosen because they are domesticated species, which are available abundantly in Malaysia and able to breed easily in captivity. In Malaysia, very little work was done on semen cryopreservation in fish species and study on fish semen cryopreservation is still at its infancy. So far only a few indigenous fish species, namely the *P. jullieni, Tor* spp, *H. nemurus, P. nasutus* and *H. wetmorei* were studied. Of which only studies on the *P. jullieni, Tor* spp., and *H. nemurus* were reported (Chew et al., 2010a; Chew et al., 2010b; Muchlisin et al., 2004), while studies on other species remain unpublished. The biology, ecology and reproduction biology of these indigenous fish species are described in the following:

#### **2.1** *Probarbus jullieni*

270 Current Frontiers in Cryopreservation

the cost and labor of maintaining broodstocks under *in situ* condition. In line with the mission of Department of Fisheries (DOF) Malaysia to develop and manage the national fisheries sector in a sustainable manner, the gene bank of freshwater fishes in the form of semen cryobank of Fisheries Research Institute at Glami Lemi was established in 2008. The establishment of the semen cryobank research has achieved the aim of the DOF towards establishing a national semen cryobank (gene bank) in Malaysia for conserving the genetic materials of the threatened or endangered indigenous freshwater fish species and also for

The main focus of this chapter will be on the methodology developed for the semen cryopreservation in Malaysia of some fish species mentioned above and the various important steps and several key factors that contributed to the successes in gamete cryopreservation. In addition, the chapter will also present the current status and the challenges of fish semen cryopreservation in Malaysia, especially on the conservation of genetic resources and potential applications of gamete cryopreservation in aquaculture. Challenges in establishment and maintenance of the fish sperm cryobank are also discussed.

Malaysia has close to a hundred river systems, two natural lakes (Lake Chini and Lake Bera) and a vast freshwater water bodies and peat swamps (Baluyut, 1983). On fish biodiversity, Malaysia has approximately 616 reported freshwater fish species (Froese & Pauly, 2003). Most of the inland fisheries resources are dominated by the cyprinids and silurids (Khoo et al., 1987). Some endemic species are found in rivers, lakes and peat swamps. In the past decades intensive development activities such as deforestation and land clearing for oil palm plantation or other agricultural uses, indiscriminate logging within and around the catchment areas and construction of dams for hydroelectricity, has led to many riverine fish species suffer high risk of extinction (Khoo et al., 1987; Jackson & Marmulla, 2001). These development activities have resulted in habitat destruction, deterioration of water quality, water pollution and sedimentation, especially during rainy season when runoff is increased. All these certainly have direct great impacts on some fish species and the impacts are irreversible (Ho, 1995). Apart from the environmental degradation resulting from development, other causes of loss in inland fisheries biodiversity are attributed to overexploitation due to strong market demand, the use of illegal and destructive fishing gears such as poisoning and electro-shocker and the introduction of invasive exotic species (Dudgeon, 2002; Allan et al*.,* 2005). The indigenous species such as Isok barb (*Probarbus jullieni*), Malaysian Mahseer (*Tor* spp.), Mad barb (*Leptobarbus hoeveni*), Hampala barb (*Hampala macrolepidota*), knife fish (*Chitala lopis*), climbing perch (*Anabas testudineus*), snakeheads (*Channa* spp.), Asian arowana (*Scleropages formosus*), the pangasiid catfishes (*Pangasius nasutus, Pangasigodon waandersii*), giant river catfish (*Wallago leerii*), large headed walking catfish (*Clarias macrocephalus*) and giant gouramy (*Osphronemus goramy*) have dwindled in great numbers continuously due to unsustainable fishing activities. At present, most Malaysian fish species could only be conserved probably in the inaccessible or remote

It is no doubt that continuing habitat destruction, overfishing and competition for food from the invading fish species are leading to loss of inland fisheries biodiversity even before much of them could be documented. The lack of data on the indigenous species will

**2. Semen cryopresevation of freshwater fish species of Malaysia** 

those indigenous species which has potential for aquaculture.

areas of the country.

*P. jullieni* (English name: Isok barb or Jullien's Golden Carp or seven-striped barb) of family Cyprinidae (Figure 1a) is commonly known as Temoleh or Temelian among Malaysians. This species is listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), Appendix 1 and the IUCN Red List as the endangered species (Hogan et al., 2009; IUCN, 2009). Therefore, conservation of this species is in urgent needed. Isok barb is a migratory species inhabiting river channels with water depth more than 10 m and is endemic to the Pahang River and Perak River in Peninsular Malaysia and the Mekong River basins of Indochina (Roberts, 1992). However, the drastic depletion of the numbers of Isok barb in Perak River was reported ever since the construction of Chenderoh Dam in 1930s. The dam created a physical barrier at the Perak River that permanently blocked the migration path of the Isok barb and increased water level further destroyed the spawning ground of this species (Khoo et al., 1987). Although the Isok barb can grow to reach the weight exceeded 70 kg, like the size of a human being (Baird, 2006), the landing of large size Isok barb (>10kg) has rarely occurred in the past 10 years.

Isok barb is a seasonal-bred species. In Malaysia, induced breeding of this species was carried out successfully in FRI Glami Lemi (formerly known as Freshwater Fisheries Research Centre)

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 273

other aquaculture species. This species usually took up to three years to reach the

The tropical bagrid catfish, *H. nemurus* (previously known as *Mystus nemurus*) of family Bagridae and with the vernacular name Baung (Figure 1d), is a widely distributed food fish found in most of the inland water bodies in Malaysia (Khan et al., 1995). The occurrence of this species in brackish water was also reported (Inger, 1955). The tropical bagrid catfish is a bottom feeder and fed on a wide range of food from small teleosts, crustaceans, benthic invertebrates to the detritus (Khan, 1987). This species is potential for aquaculture as it receives good acceptance from the local market due to its tender and non-bony meat. This carnivorous species is also a popular species in sport fishing. The artificial spawning of tropical bagrid catfish via hormonal induction using the heteroplastic pituitary extract in combination with human chorionic gonadotropin (HCG) was reported by Thalathiah et al. (1988, 1992). According to Khan et al. (1995), a bimodal male and female gonad somatic index (GSI) pattern throughout a year was shown in this species, suggesting this species

The local pangasiid catfish, *P. nasutus* of family Pangasiidae and with local name Patin Buah, (Figure 1e) is a native river catfish endemic to Pahang River in Malaysia. As commented by Hung et al. (2004), catfishes from the family Pangasiidae are of great economical importance in Southeast Asia. *P. nasutus* is one of the favorite food fish due to its white and tender flesh. This omnivorous species commands a high market value (USD 20 per kg) in local market. In the wild, *P. nasutus* fed mainly on the bivalves, mollusks, gastropods and other benthic organisms especialy on sandy bottom rivers (Roberts & Vidthayanon, 1991). Because of its economic value and declining population in the natural waters, this has created popularity and awareness to conserve and culture this species. The first successful induced spawning of *P. nasutus* was reported in 2005. This species is also a batch spawner and exhibits a single-modal GSI pattern throughout the whole year. During the spawning season, multiple releases of eggs from a single female broodfish were observed during the single period (Mohd-Zafri, 2006). The breeding season of this species is

associated with rainfalls or monsoons between May – July (Maack & George, 1999).

Morphologically *Hypsibarbus wetmorei* is quite similar to the Javanese Barb, *Barbonymus gonionotus* except the appearance of turmeric-like yellow colour on its body (Figure 1f) where the vernacular name Kerai Kunyit is given. This species is of the family Cyprinidae. Kerai Kunyit is highly regarded as a food fish and thus it is a potential aquaculture species. In Malaysia, this species is found to be endemic in Pahang River. Until today, the biology, ecology and repoductive behaviour of this species is not well studied and limited documentation on the species is available. By far, only study on species identification of *Hypsibarbus* spp. (included *H. wetmorei*) using PCR-RFLP method was reported by

marketable size of 1.5 – 2.0 kg.

**2.3** *Hemibagrus nemurus* 

could spawn twice in a year.

**2.4** *Pangasius nasutus* 

**2.5** *Hypsibarbus wetmorei*

Jantrarotai et al. (2007).

in 1990s (Ahmad-Ashhar, 1992; Ahmad-Ashhar & Haron, 1994). In its natural environment, this species spawn in fast flowing deep waters with sandy bottom. Chew et al. (2010a) reported that the spawning behavior of *P. jullieni* in captivity very much associated with the monsoons. Nevertheless, the breeding season of the cultured Isok barb was reported to be 1-2 months earlier (October to January) than the wild populations (December to February).

#### **2.2** *Tor* **spp. (***T. tambroides* **and** *T. deuronensis)*

The Malaysian Mahseer, *Tor* spp. (family: Cyprinidae) or locally known as Kelah, Empurau, Semah or Pelian, is highly sought for its value as a food fish, game fish and ornamental fish (Inger & Chin, 1962; Mohsin & Ambak, 1991; Kottelat et al., 1993). Two valid *Tor* species were identified and described in Malaysia, i.e. *T. tambroides* and *T. douronensis* (Roberts, 1989; Kottelat et al., 1993; Rainboth, 1996; Zhou & Chu, 1996) (Figure 1b and 1c). Of the two *Tor* spp., the *T. tambroides* is more favourable as food fish and can fetch up to about USD 100 – USD 220 per kg and thus is the most expensive freshwater food fish in Malaysia. Malaysian Mahseer occurs in most undisturbed and clear flowing upstream rivers, reservoir systems and lotic habitats throughout the East and Southeast Asia. The major geographical locations of Malaysian Mahseer are Pahang, Perak, Terengganu, Kelantan, Sabah and Sarawak (Ng, 2004; Ambak et al., 2007). Similar to other indigenous fishes in Malaysia, the wild populations of Malaysian Mahseer are declining over the years as the consequences of over-exploitation, natural habitat degradation and water pollution. Therefore, Malaysian Mahseer is now classified nationally as ecologically threatened (Ingram et al., 2005).

The taxonomic status of species within the genus *Tor* has been highly contentious due to plasticity of many external morphological features resulted from considerable geographical and ecological variability (Tsigenopoulos & Berrebi, 2000; Nguyen et al., 2007). Therefore, species identification based on morphological comparison sometimes could be confusing to most people who are not trained in fish taxonomy. Ability to differentiate between the Malaysian Mahseer and the Copper Mahseer (*Neolissochilus* spp.) is another difficult task hampered most of the fish farmers or aqua-culturists. The Copper Mahseer is not a desirable species and thus its value in term of market price is very much lower compared to the Malaysian Mahseer. At present, the seed supply of Malaysian Mahseer is still depending solely on the captive wild stock. As such, those who want to culture this species will have to bear with the risk of getting seed stock that comprises the mixture of these two species. Therefore, a more effective method for species identification by using DNA markers such as the mitochondria DNA (mtDNA) sequences and microsatellte markers are seen as potential in solving this problem (Nguyen et al., 2006a; Nguyen et al., 2006b; Nguyen et al., 2008; Nguyen, 2008).

The first successful hormonal induction spawning of *T. tambroides* under captive pondreared environment was reported by Ingram et al. (2005). In captive condition, *T. tambroides*  seems to spawn all year round. Brood fish of both sexes may strip up to a few times in a year. However, it is reported that geographical reproductive diversity, diet and environment conditions such as changes in temperature, water level, pH, flow velocity, turbidity, rain falls, collectively trigger the Mahseer to spawn (Dobriyal et al., 2000). Malaysian Mahseer is also a large riverine species and that can grow up to 30 – 50kg. In the culture condition, the growth rate of Malaysian Mahseer is relatively slow compared to other aquaculture species. This species usually took up to three years to reach the marketable size of 1.5 – 2.0 kg.

#### **2.3** *Hemibagrus nemurus*

272 Current Frontiers in Cryopreservation

in 1990s (Ahmad-Ashhar, 1992; Ahmad-Ashhar & Haron, 1994). In its natural environment, this species spawn in fast flowing deep waters with sandy bottom. Chew et al. (2010a) reported that the spawning behavior of *P. jullieni* in captivity very much associated with the monsoons. Nevertheless, the breeding season of the cultured Isok barb was reported to be 1-2

The Malaysian Mahseer, *Tor* spp. (family: Cyprinidae) or locally known as Kelah, Empurau, Semah or Pelian, is highly sought for its value as a food fish, game fish and ornamental fish (Inger & Chin, 1962; Mohsin & Ambak, 1991; Kottelat et al., 1993). Two valid *Tor* species were identified and described in Malaysia, i.e. *T. tambroides* and *T. douronensis* (Roberts, 1989; Kottelat et al., 1993; Rainboth, 1996; Zhou & Chu, 1996) (Figure 1b and 1c). Of the two *Tor* spp., the *T. tambroides* is more favourable as food fish and can fetch up to about USD 100 – USD 220 per kg and thus is the most expensive freshwater food fish in Malaysia. Malaysian Mahseer occurs in most undisturbed and clear flowing upstream rivers, reservoir systems and lotic habitats throughout the East and Southeast Asia. The major geographical locations of Malaysian Mahseer are Pahang, Perak, Terengganu, Kelantan, Sabah and Sarawak (Ng, 2004; Ambak et al., 2007). Similar to other indigenous fishes in Malaysia, the wild populations of Malaysian Mahseer are declining over the years as the consequences of over-exploitation, natural habitat degradation and water pollution. Therefore, Malaysian

months earlier (October to January) than the wild populations (December to February).

Mahseer is now classified nationally as ecologically threatened (Ingram et al., 2005).

The taxonomic status of species within the genus *Tor* has been highly contentious due to plasticity of many external morphological features resulted from considerable geographical and ecological variability (Tsigenopoulos & Berrebi, 2000; Nguyen et al., 2007). Therefore, species identification based on morphological comparison sometimes could be confusing to most people who are not trained in fish taxonomy. Ability to differentiate between the Malaysian Mahseer and the Copper Mahseer (*Neolissochilus* spp.) is another difficult task hampered most of the fish farmers or aqua-culturists. The Copper Mahseer is not a desirable species and thus its value in term of market price is very much lower compared to the Malaysian Mahseer. At present, the seed supply of Malaysian Mahseer is still depending solely on the captive wild stock. As such, those who want to culture this species will have to bear with the risk of getting seed stock that comprises the mixture of these two species. Therefore, a more effective method for species identification by using DNA markers such as the mitochondria DNA (mtDNA) sequences and microsatellte markers are seen as potential in solving this problem (Nguyen et al., 2006a; Nguyen et al., 2006b; Nguyen et al., 2008;

The first successful hormonal induction spawning of *T. tambroides* under captive pondreared environment was reported by Ingram et al. (2005). In captive condition, *T. tambroides*  seems to spawn all year round. Brood fish of both sexes may strip up to a few times in a year. However, it is reported that geographical reproductive diversity, diet and environment conditions such as changes in temperature, water level, pH, flow velocity, turbidity, rain falls, collectively trigger the Mahseer to spawn (Dobriyal et al., 2000). Malaysian Mahseer is also a large riverine species and that can grow up to 30 – 50kg. In the culture condition, the growth rate of Malaysian Mahseer is relatively slow compared to

**2.2** *Tor* **spp. (***T. tambroides* **and** *T. deuronensis)* 

Nguyen, 2008).

The tropical bagrid catfish, *H. nemurus* (previously known as *Mystus nemurus*) of family Bagridae and with the vernacular name Baung (Figure 1d), is a widely distributed food fish found in most of the inland water bodies in Malaysia (Khan et al., 1995). The occurrence of this species in brackish water was also reported (Inger, 1955). The tropical bagrid catfish is a bottom feeder and fed on a wide range of food from small teleosts, crustaceans, benthic invertebrates to the detritus (Khan, 1987). This species is potential for aquaculture as it receives good acceptance from the local market due to its tender and non-bony meat. This carnivorous species is also a popular species in sport fishing. The artificial spawning of tropical bagrid catfish via hormonal induction using the heteroplastic pituitary extract in combination with human chorionic gonadotropin (HCG) was reported by Thalathiah et al. (1988, 1992). According to Khan et al. (1995), a bimodal male and female gonad somatic index (GSI) pattern throughout a year was shown in this species, suggesting this species could spawn twice in a year.

#### **2.4** *Pangasius nasutus*

The local pangasiid catfish, *P. nasutus* of family Pangasiidae and with local name Patin Buah, (Figure 1e) is a native river catfish endemic to Pahang River in Malaysia. As commented by Hung et al. (2004), catfishes from the family Pangasiidae are of great economical importance in Southeast Asia. *P. nasutus* is one of the favorite food fish due to its white and tender flesh. This omnivorous species commands a high market value (USD 20 per kg) in local market. In the wild, *P. nasutus* fed mainly on the bivalves, mollusks, gastropods and other benthic organisms especialy on sandy bottom rivers (Roberts & Vidthayanon, 1991). Because of its economic value and declining population in the natural waters, this has created popularity and awareness to conserve and culture this species. The first successful induced spawning of *P. nasutus* was reported in 2005. This species is also a batch spawner and exhibits a single-modal GSI pattern throughout the whole year. During the spawning season, multiple releases of eggs from a single female broodfish were observed during the single period (Mohd-Zafri, 2006). The breeding season of this species is associated with rainfalls or monsoons between May – July (Maack & George, 1999).

#### **2.5** *Hypsibarbus wetmorei*

Morphologically *Hypsibarbus wetmorei* is quite similar to the Javanese Barb, *Barbonymus gonionotus* except the appearance of turmeric-like yellow colour on its body (Figure 1f) where the vernacular name Kerai Kunyit is given. This species is of the family Cyprinidae. Kerai Kunyit is highly regarded as a food fish and thus it is a potential aquaculture species. In Malaysia, this species is found to be endemic in Pahang River. Until today, the biology, ecology and repoductive behaviour of this species is not well studied and limited documentation on the species is available. By far, only study on species identification of *Hypsibarbus* spp. (included *H. wetmorei*) using PCR-RFLP method was reported by Jantrarotai et al. (2007).

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 275

reduced the semen quality and caused poor post-thaw sperm motility. The semen samples were then transferred back to laboratory for quantification of the fresh sperm quality and proceed with further dilution before freezing procedure. The sperm motility rates and sperm concentration of the freshly collected semen sample was evaluated prior to cryopreservation. In order to obtain good post-thaw motility, only semen samples showing

The semen samples with good quality were subsequently diluted at an appropriate sperm to diluent ratio, with suitable extender solution and cryo-protectant. Sperm to diluent ratios ranged between 1:3 to 1:9 were reported to produce best results in fish sperm cryopreservation studies (Scott & Baynes, 1980; Lahnsteiner et al., 1996). Gwo (2000) reported the fish sperm could loss its viability in high dilution ratio especially in marine species. The type of extender solution, cryoprotectant and dilution ratio that were optimum for each Malaysian fish species studied were summarized in Table 1. The type of diluents and dilution ratios workable to preserve sperm motility appear to vary among different fish species. Thus each of these

The extender solution helps to maintain sperm viability prior to and during the freezing process. Extender solution is a balanced salt buffer of specific pH and osmotic strength. Apart from salts, sometimes extender solution is prepared with addition of organic compounds such as glucose. The nature of the effect of extenders is based on the control of pH and salt concentration as well as the supply of energy, and can extend the functional life and fertilizing capability of the sperm (Tiersch, 2000). Cryoprotectants function to protect cells from cryodamage or cryo-injury during freezing and thawing process. The permeating cryoprotectants, namely dimethyl sulfoxide, methanol, ethylene glycol and propylene glycol are among the most frequently used cryoprotective agents among the aquatic organisms (Lahnsteiner et al., 1997; Tiersch, 2000; Tiersch, 2006). However, the permeating cryoprotectants are often toxic to cells, and thus the choice of the types of cryoprotectant and their optimal concentration should be at a balance between protection and toxicity. On the other hand, the non-permeating cryoprotectants such as sucrose, glucose and polymers (e.g. alginate) were often used in combination with the extender solution in the diluents. Sometimes, a combination of different cryoprotectants in certain ratios could help improving the post-thaw motility. The studies of African catfish in our laboratory has shown that a combination of methanol and N, Ndimethylacetamide at ratio 70%:30% produced significant higher post-thaw motility compared to the use of a single cryoprotectant in sample dilution. Tiersch (2000) has also reported that the application of cryoprotectant at concentration between 5% to 20% ususally provides good protection in most fish species. The protective effect and optimal concentration of cryoprotectants could be species specific (Gwo et al., 1991; Suquet et al., 2000; Rideout et al., 2003). Therefore, the optimal concentration needs to be determined individually in each

For Isok barb (*P. jullieni*), Malaysian Mahseer (*Tor* spp.), local pangasiid catfish (*P. nasutus*) and Kerai Kunyit (*H. wetmorei*), a total of 14 extender formulations, five types of cryoprotectants (dimethyl sulfoxide, ethylene glycol, glycerol, methanol and N,N dimethylacetamide) with concentrations between 5-20% (v/v), semen to diluent ratios (1:1 to 1: 14) were examined as described by Chew et al. (2010a). This may be compared with Muchlisin et al. (2004) who used three extender solutions (the Ringer, physiological saline

parameters needs to be optimized accordingly via a series of experimentations.

sperm motility >70% were used for cryopreservation.

**3.2 Dilution of semen sample** 

species studied through experimentations.

Fig. 1. Photos of the Malaysian fishes involved in the cryopreservation studies, (a) Isok barb (*P. jullieni*), (b) Malaysian Mahseer (*T. tambroides*), (c) Malaysian Mahseer (*T. deauronensis*), (d) Tropical bagrid catfish (*H. nemurus*), (e) Local pangasiid catfish (*P. nasutus*), and (f) Kerai Kunyit (*H. wetmorei*).
