**7.3 Technical limitations**

284 Current Frontiers in Cryopreservation

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.

series of complex interactions among various factors in each step.

**7.2 Maintenance and proper recording** 

**7.1 Procedure optimization** 

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

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

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

**6.2 Fish semen cryopreservation for aquaculture** 

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 considered instead.

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

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 stocks.

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

Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 287

Ahmad-Ashhar, O. (1996). Breeding and seed production technology of the Jelawat

Ahmad-Ashhar, O. (1998). Pembiakan Dan Pengeluaran Benih Ikan Jelawat (*Leptobarbus* 

Ahmad-Ashhar, O. & Haron, A. (1994). Pembiakan aruhan Ikan Temoleh *Probarbus jullieni* 

Babiak, I., Glogowski, J., Goryczko, K., Dobosz, S., Kuzminski, H., Strzezek, J. &

Baird, I. G. (2006). *Probarbus jullieni* and *Probarbus labeamajor*: the management and

Laos. *Aquatic Conservation: Marine and Freshwater Ecosystems* 16(5): 517-532. Baluyut, E. (1983). Stocking and introduction of fish in lakes and reservoirs in the ASEAN

Bart, A. (2002). Conservation of fish genetic diversity: need for development of a cryogenic

Billard, R. (1983). Effects of cleolomic and seminal fluids and various saline diluents on the

Billard, R. (1986). Spermatogenesis and spermatology of some teleost fish species. *Reprod.* 

Billard, R. & Cosson, M.P. (1992). Some problems related to the assessment of sperm motility

Billard, R., Cosson, J., Crim, L.W. & Suquet, M. (1995). Sperm physiology and quality. In:

Blaber, S.J.M., Milton, D.A., Brewer, D.T. & Salini, J.P. (2001). The shads (*genus Tenualosa*)of

Butts, I.A.E., Trippel, E.A. & Litvak, M.K. (2009). The effect of sperm to egg ratio and gamete

Chao, N.H. & Liao, I.C. (2001). Cryopreservation of finfish and shellfish gametes and

Chew, P.C., Hassan, R., Rashid, Z. A. & Chuah, H.P. (2010a). The endangered *Probarbus* 

Chew, P.C., Rashid, Z. A., Hassan, R., Asmuni, M. & Chuah, H.P. (2010b). Semen cryo-bank of the Malaysian Mahseer (*Tor* spp.) *Journal of Applied Ichthyology* 26: 726-731.

gonadotrophin (H.C.G.). *Proc. Fish. Res. Conf., DOF, Mal. IV:* 253-256. Allan, J.D., Abell, R., Hogan, Z., Revenga, C., Taylor, B.W., Welcomme, R.L. & Winemiller, K. (2005). Overfishing of Inland Waters. *BioScience* 55(12): 1041-1051. Ambak, M.A., Ashraf, A.H. & Budin, S. (2007). Conservation of the Malaysian Mahseer in

Sauvage menggunakan ekstrak pituitari dan hormon human chorionic

Demianowicz, W. (2001). Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved

conservation of two of the largest fish species in the Mekong River in southern

genebank in Bangladesh. In: Penman, D.J., Hussain, M.G., McAndrew, B.J. and Mazid, M.A. (eds.) Proceedings of a workshop on Genetic Management and Improvement Strateges for Exotic Carps in Asia, 12-14 February 2002. Dhaka,

fertilizing ability of spermatozoa in the Rainbow trout, *Salmo gairdneri. J. Reprod.* 

*Brood stock management and egg and larval quality.* (Eds) Bromage, N. R. and Roberts,

tropical Asia: An overview of their biology, status and fisheries. In: *Proceedings of the International Terubok Conference Sarawak, Malaysia, 11 -12 September 2001, Sarawak.* 

contact time on fertilization success in Atlantic cod *Gadus morhua* L. *Aquaculture* 286

*jullieni* (Sauvage) - Current status on its sperm cryopreservation in Malaysia.

(*Leptobarbus hoevenii*).

*hoevenii*) Bleeker. *Buku Panduan Bil 2/98*. 21pp.

Nenggiri basin through community action.

spermatozoa. *Theriogenology* 56: 177–192.

in freshwater fish. *J. Exp. Zool.*, 261: 122-131.

R.J. Blackwell Science, Oxford. pp. 25-52.

embryos. *Aquaculture* 197: 161-189.

*Journal of Applied Ichthyology* 26: 797-805.

region. *FAO Tech. Pap.* 236. 82 pp.

Bangladesh. Pp 107-110.

*Nutr. Dev.,* 2: 877-920.

*Ferti.,* 68: 77-84.

pp. 9-17.

(1-2):89 – 94.

enable species to adapt to environmental change and also provide the opportunity for genetic improvement programme in aquaculture.

It is observed that large species that breed later in life are more vulnerable to fishing and changes in the environment, particularly in terms of fragmentation of their normal habitats. Indeed, most of the world's largest freshwater fish are at risk according to the IUCN Red List, and over exploitation contributes in a number of these cases. The dragon fish, *Scleropages formosus* is a well known case of over-exploitation. Therefore, giant indigenous species such as freshwater siluroids (*Wallago leeri,*), cyprinids (*Tor* spp., *Probarbus jullieni*), pangasiids (*Helicophagus waandersii*) etc. can be promoted as 'flagship species' or ecosystem ambassadors. At the same time, in terms of preserving biodiversity, by reducing the negative impacts of the continued spread of exotic fish species in the aquatic environment, efforts need to develop indigenous species for use in aquaculture. Those indigenous species that showed good adaptation to pond environment, resistance to handling, possess high growth rate and ability to reach sexually maturity in captivity are worth to be considered and developed as aquaculture species. To achieve the goal, we must safeguard indigenous fish resources both quantitatively and qualitatively from now before it is too late.
