**1. Introduction**

268 Current Frontiers in Cryopreservation

Rana, K.L. (1995). Preservation of Gametes. In: *Broodstock Management and Egg and Larval* 

Szabó, T. (2000). A csuka tógazdasági tenyésztése In: *Halbiológia és haltenyésztés*, L. Horváth,

Urbanyi, B.; Horváth, Á. & Dinnyés, A. (1999). Cryopreservation of African catfish (*Clarias* 

Urbányi, B.; Szabó, T.; Miskolczi, E.; Mihálffy, Sz.; Vranovics, K. & Horváth, Á. (2006).

Wheeler P.A. & Thorgaard G.H., 1991: Cryopreservation of rainbow trout semen in large

straws. *Aquaculture,* Vol.93, pp. 95–100, ISSN: 0044-8486

03591-5, Stirling, UK

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pp. 201-204, ISSN: 0175-8659

*Quality,* N.R. Bromage & R.J. Roberts, (Eds.), 53-75, Blackwell, ISBN 978-0-632-

(Ed.), 312 Mezőgazda Kiadó, ISBN 963-9239-45-3, Budapest, Hungary (in

*gariepinus*) sperm using different cryoprotectants. *Theriogenology*, Vol.51, p. 296,

Successful fertilization and hatching of four European cyprinid species using cryopreserved sperm. *Journal of Applied Ichthyology*, Vol.22,

> It has been estimated that spermatozoa can last from 200-32,000 years (Stoss & Donaldson, 1983; Suquet et al*.,* 2000). According to Kopeika et al. (2007), several methods of fish sperm storage has been practised including stored in medium with saturated gases, preservation at temperature above zero, in a frozen state as well as by drying. However, the low temperature approaches have been successful in fish sperm cryopreservation. Thus, cryopreservation technology offers the best means for long term storage of fish semen.

> To date, successful cryopreservation of fish semen were reported in more than 200 freshwater species and 40 marine species worldwide (Gwo, 2000). Even though in general many successes have been achieved in fish semen cryopreservation, the technique remains as a method that is difficult to be standardized and use in all types of fishes. This is due to the fact that cryopreservation of sperms from different fish species required different conditions, where the protocol needs to be established individually. Even the "general protocol" of cryopreservation of fish sperm summarized by Kopeika et al. (2007) encompassed many variations when different species of fish are involved, particularly in the use of medium ingredients for the cryopreservation.

> In view of need to develop individual protocol for successful cryopreservation of fish semen and considering Malaysia has a rich fish fauna with many of them unique to this tropical region, cryopreservation of fish gametes will require detailed study to create new protocol for each fish species intended for semen cryopreservation. To date in Malaysia semen cryopreservation has only been reported for several freshwater fish species, namely *Probarbus jullieni, Tor tambroides, T. deuronensis*, *Hemibagrus nemurus*, *Pangasius nasutus, Hypsibarbus wetmorei, Barbonymus gonionotus* and *Clarias gariepinus.* It has been demonstrated that semen cryoperservation plays an important role for the genetic conservation of these fish species.

> Cryopreservation technology for fish semen is still not well explored in Malaysia and can be considered as new if compared to the domesticated terrestrial livestocks. Henceforth, this has opened up a new field to be explored with potential applications in aquaculture and in the conservation of the national fisheries genetic resources. Cryopreserved semen could facilitate artificial fertilization especially when mature male fishes are not available or unable to provide viable semen during certain periods of the breeding season. Semen cryopreservation may also be useful for fertilization to produce hybrids of various fish species. It also helps in reducing

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 271

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;

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:

large size Isok barb (>10kg) has rarely occurred in the past 10 years.

*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

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)

Suhairi, 1996; Suhairi et al., 1996; Ahmad-Ashhar, 1998).

**2.1** *Probarbus jullieni* 

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 those indigenous species which has potential for aquaculture.

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.
