**8. Conclusion**

An overview of the current status of semen cryopreservation of Malaysian freshwater fishes is presented in this chapter. The role of semen cryobank is also discussed. Obviously, semen cryopreservation offers potential applications in *ex situ* conservation and sustainable management of the fisheries genetic resources in Malaysia, especially for those species with rare, vulnerable, threatened or endangered status, those protandry species with sexchanging characteristic over their life time, and also the potential candidates and genetic improved strains for aquaculture development. The successes in semen cryopreservation are very much relied on factors such as having ample knowledge on the biology and reproductive biology of the particular species of interest, trained personnels in various aspects such as gamete cryopreservation, breeding methods, broodstock management and husbandry and larva rearing and nursing of the targeted species.

### **9. Acknowledgement**

We would like to thank the Department of Fisheries Malaysia for supporting this research through a development grant and also thanks to all research staffs of Freshwater Fisheries Research Division, FRI Glami Lemi and the Aquaculture Extension Centre, Perlok who have kindly provided assistance during the project.

#### **10. References**


enable species to adapt to environmental change and also provide the opportunity for

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.

husbandry and larva rearing and nursing of the targeted species.

An overview of the current status of semen cryopreservation of Malaysian freshwater fishes is presented in this chapter. The role of semen cryobank is also discussed. Obviously, semen cryopreservation offers potential applications in *ex situ* conservation and sustainable management of the fisheries genetic resources in Malaysia, especially for those species with rare, vulnerable, threatened or endangered status, those protandry species with sexchanging characteristic over their life time, and also the potential candidates and genetic improved strains for aquaculture development. The successes in semen cryopreservation are very much relied on factors such as having ample knowledge on the biology and reproductive biology of the particular species of interest, trained personnels in various aspects such as gamete cryopreservation, breeding methods, broodstock management and

We would like to thank the Department of Fisheries Malaysia for supporting this research through a development grant and also thanks to all research staffs of Freshwater Fisheries Research Division, FRI Glami Lemi and the Aquaculture Extension Centre, Perlok who have

Aas, G.H.; Refstie, T. & Gjerde, B. (1991). Evaluation of milt quality of Atlantic salmon.

Ahmad-Ashhar, O. (1992). Induced spawning of Ikan Temoleh *Probarbus jullieni* Sauvage

using human chrionic gonadotropin and heteroplastic crude piscine extract. In: Proceedings of the National IRPA Seminar, Kuala Lumpur, 9-11 January 1992. pp.

genetic improvement programme in aquaculture.

**8. Conclusion** 

**9. Acknowledgement** 

**10. References** 

243-244.

kindly provided assistance during the project.

Aquaculture, 95: 125-132.


Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 289

Hung, L. T., Suhenda, N., Slembrouck, J., Lazard, J. & Moreau, Y. (2004). Comparison of

Inger, R.F. (1955). Ecological notes on the fish fauna of a coastal drainage of north Borneo.

Inger, R.F. & Chin, P.K. (1962). The Freshwater Fishes of North Borneo. *Fieldiana Zool.* 45: 1-

Inger, R.F. & Chin, P.K. (2002). *The Freshwater Fishes of North Borneo with a supplementary* 

Ingram, B., Sungan, S., Gooley, G., Sim, S.Y., Tinggi D. & de Silva S.S. (2005). Induced

IUCN (2009). IUCN Red List of Threatened Species. Version 2009.1. <www.iucnredlist.org>.

Jackson, D.C. & Marmulla, G. (2001). The influence of dams on river fisheries. In Dams, fish

Jantrarotai, P., Sutthiwises, S., Kamonrat, V., Peyachoknagul, S. & Vidthayanon, C. (2007).

Khan, M.S. (1987). Some aspects of the biology of ikan baung, *Mystus nemurus* C. & V. with

Khan, M.S., Ambak, M.A., Ang, K.J. & Mohsin, A.K.M. (1995). Reproductive biology of a

Khoo, K.H., Leong, T.S., Soon, F.L., Tan, S.P. & Wong, S.Y. (1987). Riverine fisheries in

Kincaid, H.L. (2000). Development of Databases for Germplasm Repositories. In: Tiersch,

Kopeika, E., Kopeika, J. & Zhang, T. (2007). Cryopreservation of fish sperm. In: Day, J.G. and

Kottelat, M., Whitten, A.J., Kartokasari, S.N. & Wirjoratmodjo, S. (1993). *Freshwater Fishes of* 

Kurokura, H., Hirano, R., Tomita, M. & Iwahashi, M. (1984). Cryopreservation of carp

Lahnsteiner, F. & Patzner, R.A. (2008). Sperm morphology and ultrastructure in fish. In:

Lahnsteiner, F., Patzner, R.A. & Weismann, T. (1996). Semen cryopreservation of salmonid

Lahnsteiner, F., Weismann, T. & Patzner, A.R. (1997). Methanol as cryoprotectant and the

*Protocols, 2nd edition.* Humana Press Inc., Totowa, NJ. pp. 203 -217.

*Western Indonesia and Sulawesi*. Singapore: Berkeley Book Pte Ltd.

*Tor tambroides* and *T. duoronensis*. *Aquacult. Res*. 36(10): 1001-1014.

*hypophthalmus* and *P. djambal*). *Aquaculture Nutrition* 10: 317-326.

*Fieldiana Zool.* 37: 47 -90.

Downloaded on 25 August 2009.

Society, Baton Rouge.Pp 323-331.

sperm. *Aquaculture* 37: 267-273.

Aquacult. Res. 27(9): 659–671.

fishes. *Aquaculture Research* 28: 471-479.

Science International Ltd., Oxford, UK. pp. 1-61.

*FAO Fisheries Technical Paper* 419: 1-45*.* 

cytochrome b gene. *Kasetsart J. (nat. Sci.)* 4: 660 – 666.

Malaysia. *Aquaculture and Fisheries Management* 21: 173-179.

Malaysia. *Archiv. feur Hydrobiologie Beiheft*. 28: 261 – 268.

268.

268 pp. + S-78.

Serdang.

dietary protein and energy utilization in three Asian catfishes (*Pangasius bocourti, P.* 

*chapter by Chin Phui Kong*. Natural History Publications (Borneo), Kota Kinabalu.

spawning, larval development and rearing of two indigenous Malaysian Mahseer,

and fisheries: Opportunities, challenges and conflict resolution (ed. Marmulla, G.)*.* 

Species identification of 3 *Hypsibarbus* spp. (Pisces: Cyprinidae) using PCR-RFLP of

reference to Chenderoh Reservoir. Thesis of MSc. Universiti Pertanian Malaysia,

tropical catfish, *Mystus nemurus* Cuvier and Valenciennes, in Chenderoh Reservoir

T.R. and Mazik, P.M. (eds) *Cryopreservation in Aquatic Species.* World Aquaculture

Stacey, G.N. (Eds) *Methods Mol. Biol. Vol.* 368: *Cryopreservation and Freeze-Drying* 

Alavi, S.M.H., Cosson, J.J., Coward, K. and Rafiee, G. (Eds) *Fish Spermatology*. Alpha

fishes: influence of handling parameters on the post thaw fertilization rate,

suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid


Chong, V.C., Lee, P.K.Y. & Lau, C.M. (2010). Diversity, extinction risk and conservation of

Curry, M.R. (2000). Cyopreservation of semen from domestic livestock. *Reviews of* 

Dobriyal, A.K., Kumar, N., Bahuguna, A.K. & Singh, H.R. (2000). Breeding ecology of some

DOF. (n.d.). Endangered freshwater fish species in Malaysia under the IUCN Red List. In: *3rd*

Donoghue, A.M. & Wishart, G.J. (2000). Storage of poultry semen. *Animal Reproduction* 

Dreanno, C., Suquet, M., Quemener, L., Cosson, J., Fierville, F., Normant, Y. & Billard, R.

Dudgeon, D. (2002). Fisheries : pollution and habitat degradation in tropical Asian rivers.

FAO. (1971). Seminar/Study Tour in the U.S.S.R. on Genetic Selection and Hybridization of

Froese, R., & Pauly, D. (2003). (Eds.). *Fishbase*. In: *World Wide Web electronic publication, 3rd*

Gwo, J.C. (2000). Cryopreservation of sperm of some marine fishes. In Tiersch, T.R. and

Gwo, J.C., Strawn, K., Longnecker, M.T. & Arnold, C.R. (1991). Cryopreservation of Atlantic

Hammerstedt, R.H. & Graham, J.K. (1992). Cryopreservation of poultry sperm: the enigma

Harvey, B. (1998). An overview of action before extinction. In: Harvey B, Ross C, Greer D,

Ho, S.C. (1995). Status of Limnological Research and Training in Malaysia. In: Limnology in

Hogan, Z., Baird, I.G. & Phanara, T. (2009). Threatened fishes of the world: *Probarbus jullieni*  Sauvage, 1880 (Cypriniformes: Cyprinidae). *Environ. Biol. Fish* 84: 291-292. Honeyfield, D.C. & Krise, W.F. (2000). Measuament of milt quality and factors affecting

Horvath, A., Miskolczi, E. & Urbányi, B. (2003). Cryopreservation of common carp sperm.

*January 2011. Available from: <http://www.fishbase.org>.* 

croakker spermatozoa. *Aquaculture* 94: 355-375.

*<http://www.dof.gov.my/c/document\_library/get\_file?uuid=d8ebcbc5-4d4a-4125-8290-*

(1997). Cryopreservation of turbot (*Scophthalmus maximus*) spermatozoa.

*Encyclopaedia of Global Environmental Change, Vol. III* (ed. I. Douglas), pp. 316–323.

Cultivated Fishes. 19 April – 29 May 1968. Lectures. Rep.FAO/UNDP(TA), (2926):

Mazik, P.M. (Eds.), *Cryopreservation in Aquatic Species*. World Aquaculture Society,

Carolsfeld J (eds) Action before extinction: an international conference on conservation of fish genetic diversity. World Fisheries Trust, Victoria Inger, F.R. and Chin, P.K. 1962. *The Freshwater Fishes of North Borneo*. Chicago Natural History

Developing Countries (eds B. Gopal and R.G. Wetzel). International Association for Limnology. International Scientific Publications, 50-B., Pocket C., Sidhartha

viability of fish spermatozoa. In: Tiersch, T.R. and Mazik, P.M. (Eds.), *Cryopreservation in Aquatic Species*. World Aquaculture Society, Baton Rouge, L.A.

cold water minor carps from Garhwal Himalayas. Coldwater Fish and Fisheries: 177-186. (Eds) Singh, H.R. and Lakra, W.S. Narenda Publishing House, New Delhi.

Malaysian fishes. *Journal of Fish Biology* 76: 2009-2066.

*Reproduction* 5: 46-52.

*January 2011. Available from:* 

*Theriogenology* 48: 589-603.

*Science* 62: 213-232.

*5d5bd78b1a5c&groupId=172176>.*

John Wiley & Sons, Chichester, U.K.

Baton Rouge, L.A. pp. 138-160.

of glycerol. *Cryobiology* 29: 26-38.

Extension, New Delhi. Pp 163-189.

*Aquat. Living Resour.* 16: 457–460.

Muzeum, Chicago.

pp. 49-58.

337p.

360 p.


Sperm Cryopreservation of Some Freshwater Fish Species in Malaysia 291

Nguyen, T.T.T., Ingram, B., Sungan, S., Gooley, G., Sim, S.Y., Tinggi, D. & De Silva, S.S.

Nguyen, T.T.T., Ingram, B., Sungan, S., Tinggi, D., Sim, S.Y., Gooley, G. & De Silva, S. S.

Nguyen, T.T.T., Sukmanomon, S., Na-Nakorn, U. & Ziming, C. (2008). Molecular phylogeny

Pathmasothy, S. (1985). The Effect of Three Diets with Variable Protein Level on Ovary

Pathmasothy, S., & R. Omar. (1982). The Effect of Four Different Diets on the Growth of

Rainboth, W.J. (1996). *Fishes of the Cambodian Mekong. FAO Species Identification Field Guide for Fishery Purposes.* Food and Agriculture Organization (FAO) Publication, Rome. Rideout, R.M., Litvak, M.K. & Trippel, E.A. (2003). The development of a sperm

Roberts, T.R. (1989). *The Freshwater Fishes of Western Borneo (Kalimantan Barat,* 

Roberts, T.R. (1992). Revision of the Southeast Asian cyprinid fish genus *Probarbus*, with two

Roberts, T.R. & Vidthayanon, C. (1991). Systematic revision of the Asian catfish family

Scott, A.P. & Baynes, S.M. (1980). A review of the biology, handling and storage of salmonid

Stoss, J & Donaldson, E.M. (1983). Studies on cryopreservation of eggs from rainbow trout (*Salmo gairdneri*) and coho salmon (*Oncorhynchus kisutch*). *Aquaculture* 31: 51 – 65. Suhairi, A. (1996). Preliminary observations on the culture of the Temoleh (*Probarbus jullieni*)

Suhairi, A., Misri, S., Abdul Ghani, H. & Ahmad Ashhar, O. (1996). Kajian awal ternakan

Suquet, M., Omnes, M.H., Normant, Y. & Fauvel, C. (1992). Assessment of sperm

*Proceedings of the Academy of NaturalSciences of Philadelphia* 143: 97-144. Saidin, T. (1986). Induced spawning of *Clarias macrocephalus* (Gunther). In: Maclean, J.L.,

species in Sarawak, Malaysia. (Downloaded on 20 October 2009)

*Evolution* (in press) (doi:10.1016/j.ympev.2008.01.006).

Singapore 23-2 August, 1983. Ottawa: IDRC., pp.107-112.

*Indonesia).*California Academy of Sciences, California.

River. *Ichthyl. Expl. Freshw.* 3: 37-48.

Society, Manila, Philipines. P. 683 – 686*.*

spermatozoa, J. Fish Biol. 17: 707–739.

Pinang, Malaysia.

*1996*: 278-283.

185.

*Leptobarbus hoevenii*. *MARDI Research Bulletin* 10(1): 100-113.

*Aquaculture* 253: 259-269.

653-659.

(2006). Mitochondrial diversity of broodstock of two indigenous mahseer species, *Tor tambroides* and *T. douronensis* (Cyprinidae) cultured in Sarawak, Malaysia.

(2007). Guidelines for genetic management and conservation of two mahseer

and phylogeography of mahseer species (Cyprinidae: Cypriniformes) as inferred from sequences of two mitochondrial gene regions. *Molecular Phylogenetics and* 

Development and Fecundity in *Leptobarbus hoevenii*. In: Cho, C.Y., D.S. Cowey and W. Watanabe, Finfish Nutrition in Asia; Methodological Approach to Research and Development. Proceedings of the Asian Finfish Nutrition Workshop Held in

cryopreservation protocol for winter flounder, *Pseudopie-uronectes americanus*  (Walbaum): evaluation of cryoprotectants and diluents. *Aquaculture Research* 34:

new species threatened by the proposed construction of dams on the Mekong

Pangasiidae, with biological observations and description of three new species.

Dizon, L.B. & Hosillos, L.V. (eds.) *The First Asian Fisheries Forum.* Asian Fisheries

in ponds. Paper presented in *Fisheries Semeniar July 1996*, IPP Batu Maung, Pulau

ikan temoleh (*Probarbus jullieni*) dalam kolam. *Proc. Fish. Res. Conf., DOF, Mal.,* 

concentration and motility in turbot *Scophthalmus maximus. Aquaculture* 101: 177-


Lahnsteiner, F., Berger, B. & Weismann, T. (2003). Effects of media, fertilization technique,

Leung, L.K.P. & Jamieson, B.G.M. (1991).Live preservation of fish gametes. In: Jamieson,

Maack, G.G. & George, M.R. (1999). Contributions to the reproductive biology of

MACD (1996). Life Cyle of Terubok (*Tenalosa toli*) With Recommendations for the

Mohd-Zafri, H. (2006). Morphology and general reproductive stages of *Pangasius nasutus*  from Sg. Pahang in Maran District, Pahang, Malaysia. Thesis Msc, UPM. Mohsin, A.K.M. & Ambak, M.A. (1991). *Freshwater Fishes of Peninsular Malaysia.* Penerbit

Mongkonpunya, K., Pupipat, T. & Tiersch, T.R. (2000). Cryopreservation of sperm of Asian

Morisawa, M. & Suzuki, K. (1980). Osmolality and potassium ion: Their roles in initiation of

Morisawa, M., Suzuki, K. & Morisawa, S. (1983a). Effects of potassium and osmolality on

Morisawa, M., Suzuki, K., Shimizu, H., Morisawa, S. & Yasuda, K. (1983b). Effects of

Muchlisin, Z.A., Hashim, R. & Chong, A.S.C. (2004). Preliminary study on the

Munkittrick, K.R. & Moccia, R.D. (1984). Advances in the cryopreservation of salmonid

Ng, C.K. (2004). *Kings of the Rivers – Mahseer in Malaysia and the region.* Inter Sea Fishery (M)

Nguyen, T.T.T. (2008). Population structure in the highly fragmented range of *Tor* 

Nguyen, T.T.T., Baranski, M., Rourke, M. & McPartlan, H. (2006). Characterisation of

Catfishes including the endangered Mekong Giant Catfish. In: Tiersch, T.R. and Mazik, P.M. (Eds.), *Cryopreservation in Aquatic Species*. World Aquaculture Society,

spermatozoan motility of Salmonid fishes. *Journal of Experimental Biology* 107: 105 –

potassium and osmolality on motility of spermatozoa from freshwater cyprinid

cryopreservation of tropical bagrid catfish (*Mystus nemurus*) spermatozoa: the effect of extender and cryoprotectant on the motility after short-term storage.

semen and suitability for a production-scale artificial fertilization program.

*douronensis* (Cyprinidae) in Sarawak, Malaysia revealed by microsatellite DNA

microsatellite DNA markers for the mahseer species, *Tor tambroides* and crossamplification in other four congeners (*T. douronensis, T. khudree, T. putitora,* and *T.* 

Agriculture & Community Development (MACD), Kuching. Pp.64. Mohamad-Zaini S. (1992). Pembiakan Aruhan Dan Pengeluaran Benih Ikan Sebarau

*Hampala macrolepidota*. *Buletin Perikanan* 82. 13 pp.

Universiti Pertanian Malaysia, Serdang, Selangor.

sperm motility in eleosts. *Science* 210: 1145 – 1147.

fishes. *Journal of Experimental Biology* 107: 95 – 103.

embryos, using cryopreserved semen. *Theriogenology* 60: 829-841.

Univ. Press, Cambridge, pp. 245–269.

*Fisheries Research* 44: 113 – 120.

Baton Rouge, L.A. pp. 108-116.

*Theriogenology* 62: 25-34.

*Theriogenology* 21: 645-59.

markers. *Freshwater Biology* 53: 924-934.

*tor*). *Molecular Ecology Notes* 7: 109-112.

Sdn. Bhd. Selangor.

113.

extender, straw volume, and sperm to egg ratio on hatchability of cyprinid

B.G.M. (Ed.), *Fish Evolution and Systematics: Evidence from Spermatozoa.* Cambridge

*Encrasicholina punctifer* Fowler, 1938 (engraulidae) from West Sumatra, Indonesia.

Conservation, Management and Culture of The Species. Phase 3 Final Report. A collaborative project of CSIRO Division of Fisheries, Marine Laboratories, Cleveland Queensland, Australia and Inland Fisheries Branch, Ministry of


**15**

*Turkey* 

**Cryopreservation of Brown Trout (***Salmo trutta* 

*1Mustafa Kemal University, Faculty of Fisheries, Department of Aquaculture, Hatay 2Mustafa Kemal University, Faculty of Veterinary Medicine, Department of Reproduction* 

Cryopreservation is considered as one component in an effective strategy to save endangered species by facilitating the storage of their gametes in gene banks (Gausen, 1993; Akcay et al. 2004). Cryopreservation offers several benefits that in this way stocks can be protected from being totally eliminated due to sudden disease outbreak, natural utilization in hatcheries' production and laboratory experiments can be ensured. Stocks can be maintained more economically and experimental materials for advanced studies, such as

Cryopreservation techniques involve addition of cryoprotectants, freezing and thawing of sperm samples, all of which may result in some damage to the spermatozoa and may decrease egg fertilization rate (Kopeika et al. 2003). Therefore, before cryopreservation of sperm, a through evaluation of different extender solutions, cryoprotectants, straw sizes and thawing rates is essential to develop optimum cryopreservation protocols for various

The species-specific cryopreservation procedure needs a suitable extender, as undiluted semen is not suitable for long-term preservation. Similarly, addition of optimum amount of cryoprotectant reduces cell damages associated with dehydration, cellular injuries and ice crystal formation (Leung, 1991). Although cryoprotectants help to prevent cryoinjuries during freezing and thawing, they can become toxic to cells when the exposure time and concentration are more (Tekin et al. 2007). In addition, type of cryoprotectants is also very specific to many species. There are no universal extenders and cryoprotectants available that

On the other hand, motility is the most commonly used parameter to evaluate sperm quality in fishes (Billard et al. 1995). This parameter is acceptable so that spermatozoa must be motile to achieve fertilization. Furthermore, sperm motility is an important component of a cryopreservation program in order to prevent poor sperm quality semen samples prior to freezing and to estimate the fertility of the stored sperm after thawing (Akçay et al., 2004;

gene transfer, can be made more accessible (Chao & Liao, 2001; Tekin et al. 2003).

**1. Introduction** 

species (Yavas & Bozkurt, 2011).

can be used across species.

*macrostigma***) and Ornamental Koi Carp** 

**(***Cyprinus carpio***) Sperm** 

*and Artificial Insemination, Hatay* 

Yusuf Bozkurt1, İlker Yavas2 and Fikret Karaca2

