**1. Introduction**

304 Current Frontiers in Cryopreservation

Leung, L.K.P. (1991). Principles of biological cryopreservation. In. *Fish evolution and* 

Linhart O.; Rodina M. & Cosson, J. (2000). Cryopreservation of sperm in common carp: sperm motility and hatching success of embryos. *Cryobiology*, 41:241-250. Mansour, N.; Richardson, G.F. & McNiven, M.A. (2006). Effect of extender composition and

Morisawa, S. & Morisawa, M. (1986). Acquisition of potential for sperm motility in rainbow trout and chum samon. *The Journal of Experimental Biology*, 126: 89-96. Morris, G.J. (1981). *Cryopreservation: an introduction to cryopreservation in culture collections.*

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

Rana, K.J. & McAndrew, B.J. (1989). The viability of cryopreserved tilapia spermatozoa,

Ravinder, K.; Nasaruddin, K.; Majumdar, K. C. & Shivaji, S. (1997). Computerized analysis

Rurangwa, E.; Volckaert, F.A.M.; Huyskens, G.; Kime, D.E. & Ollevier, F. (2001). Quality

Secer, S.; Tekin, N.; Bozkurt, Y.; Bukan, N. & Akçay, E. (2004). Correlation between

Stoss, J. & Holtz, W. (1983). Successful storage of chilled rainbow trout (*Salmo gairdneri*)

Tekin, N.; Secer, S.; Akcay, E. & Bozkurt, Y. (2003). Cryopreservation of rainbow trout

Tekin, N.; Secer, S; Akcay, E.; Bozkurt, Y. & Kayam, S. (2007). Effects of glycerol additions on

Vuthiphandchai, V. & Zohar, Y. (1999). Age-related sperm quality of captive striped bass,

Wayman, W.R.; Tiersch, T.R. & Thomas, W.R. (1998). Refrigerated storage and

Yavas, I. & Bozkurt, Y. (2011). Effect of different thawing rates on motility and fertilizing

*Morone saxatilis*. *Journal of World Aquaculture Society*, 30: 65–72.

*and Biotechnological Equipment*, 25 (1), 2254-2257.

*mykiss*) semen. *The Israeli Journal of Aquaculture-Bamidgeh*, 56 (4), 274-280. Steyn, G.J. & Van Vuren, J.H.J. (1987). The fertilising capacity of cryopreserved sharptooth

(L.), spermatozoa. *Aquaculture Research*, 37: 862– 868.

Cambridge (England): Institute of Terrestrial Ecology.

storage of semen. *Journal of Fish Biology*, 50: 1309–1328.

cattish (*Clarias gariepinus*) sperm. *Aquaculture*, 63: 187-193.

spermatozoa for up to 34 days, *Aquaculture*, 31: 269–274.

(*Clarias gariepinus*). *Theriogenology*, 55:751-769.

Press, Cambridge. 231-244.

*Theriogenology*, 62, 25–34.

*Aquaculture*, 76: 335-345.

212.

*Society*, 127: 95-104.

29: 267-273.

*systematics: Evidence from spermatozoa*. Jamieson, B.M.G. (Ed). Cambridge University

freezing rate on post-thaw motility and fertility of Arctic char, *Salvelinus alpinus* 

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

of motility, motility patterns and motility parameters of carp following short-term

control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilisation in African catfish

biochemical and spermatological parameters in rainbow trout (*Oncorhynchus* 

(*Oncorhynchus mykiss*) semen. *The Israeli Journal of Aquaculture-Bamidgeh,* 55 (3), 208-

post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant. *Journal of Applied Ichthyology*, 23 (1): 60-63. Tiersch, T.R.; Williamson, J.H.; Carmichael, G.J. & Gorman, O.T. (1998). Cryopreservation of

sperm of the endangered razorback sucker. *Transactions of the American Fisheries* 

cryopreservation of sperm of red drum, *Sciaenops ocellatus* L. *Aquaculture Research*,

capacity of cryopreserved grass carp (*Ctenopharyngodon idella*) sperm. *Biotechnology* 

The production of fish in Nigeria is still very small and cannot sufficiently satisfy the increasing demand of its population of 140 million. To solve the populace's high demand for fish, Nigerians resort to aquaculture which is currently faced with major constraints including lack of fish seed and quality of feed. The scarcity of good broodstock has necessitated the need to conserve the fish genetic resources which are wasted during natural and artificial induced spawning process of fish breeding. One way of expanding aquaculture in Nigeria is by devising a means of preserving genetic resources of our broodstock for all year round supply of fish seed through cryopreservation (Omitogun *et al*., 2006).

The African catfish *Clarias gariepinus* Burchell, 1822 is one of the most suitable species for aquaculture in Africa. Since the 1970's, it has been considered to hold a great promise for fish farming in Africa. Some other merits of African catfish are: high growth rate reaching market size of 1 kg in 5–6 months under intensive management conditions: highly adaptable and resistant to handling and stress; can be artificially propagated by induced spawning techniques for reliable mass supply of fingerlings; commands a very high commercial value where it is highly cherished as food in Nigerian homes and hotels (Olaleye, 2005.).

The Clariid freshwater fishes belong to the family Clariidae with a wide geographical distribution in Africa consisting of 14 genera (Teugels, 1986a) and 32 species (Teugels, 1986b) in Nigeria. Syndenham (1980) reported that the family consists of 5 subgenera namely: Clarias, Clarioides, Anguilloclarias, Platycephaceloides, and Brevicephaloides*. C.* 

<sup>\*</sup> Corresponding Author

Cryopreservation of the Sperm

in the dry season.

**eggs** 

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 307

commercial scale and is proven economically feasible, being a true reflection of what was obtained in the laboratory (Oyeleye and Omitogun, 2007) then this will help to conserve male brood stock (Omitogun *et al.*, 2010) which are normally slaughtered for fry production of catfish, and likewise ensure all-year round artificial propagation, helping the fish farmers in overcoming the problem of scarcity of male catfish breeders which are often encountered

**1.2 Background information: Sperm: Egg ratio for optimum fertilization of catfish** 

meet high demand for catfish consumption (Oyeleye and Omitogun, 2007).

egg to hatching is dependent upon water temperature (Coppens, 2009).

of protocols difficult (Viveiros *et al.*, 2000).

Cryopreservation of African catfish semen in liquid Nitrogen (LN2) will invariably help us to conserve the genetic resources of our desirable male fish breeders for all year round artificial propagation and also help in overcoming the problems of scarcity of desirable male catfish breeders often encountered by the farmers most especially in the dry season and to

Sperm collection in African catfish as mentioned requires killing the male fish in order to excise the testes, it is important to maximize the use of a single male by optimization of sperm: egg insemination ratio. For fresh spermatozoa, the effective insemination ratio was estimated as 245 x 103 spermatozoa per egg in *C. gariepinus (*Steyn, 1987) and 50 x 103 spermatozoa per egg in *Heterobranchus longifilis* (Otenne *et al.,* 1996.)*.* Because a percentage of spermatozoa die during freezing and thawing processes, the effective insemination ratio for frozen spermatozoa should be higher. In channel catfish, 50 x 106 frozen-thawed spermatozoa per 0.5 ml straw enabled fertilization of 250 eggs (200 x 103 spermatozoa per egg; Tiersch *et al.,* 1994). In blue catfish, *Ictalurus furcatus*, a minimum of 13,000 x 103 frozenthawed spermatozoa per egg were needed to achieve 54% of control fertilization. In *C. gariepinus*, 49 x 103 live frozen-thawed spermatozoa per egg achieved a hatching rate (51.2%) equal to the control (51%).The insemination ratio was within the range 6 to 24 x 103 spermatozoa per egg. (Steyn, 1993). During ovulation the belly of the female will swell considerably due to water absorption of the ovary. The speed of the ripening process is dependent upon water temperature and likewise, the development process from fertilized

African catfish spermatozoa were first successfully cryopreserved by Steyn *et al.* 1985 who obtained 40% motility 24h after storage in LN2. Glucose in combination with glycerol has been most widely used cryoprotective solution. Recently, glucose in combination with DMSO was also shown to be effective (Urbanyi *et al.*, 1999). Freezing rates can be rapid (*e.g*., pellet freezing on dry ice or in LN2 vapor) or slow (*e.g*., at fixed rates in programmable freezer (Steyn, 1993). In most cases, sperm quality was only evaluated in terms of motility after thawing. When fertilization was included in the evaluation, sperm: egg ratio was not optimized and was often excessive (Padhi *et al.*,1995). Using excess spermatozoa for fertilization obviously masks the quality of cryopreserved spermatozoa, making comparison

Methods for cryopreserving spermatozoa and optimizing sperm: egg dilution ratio in African catfish *Clarias gariepinus* was first developed by Viveiros *et al*., 2000) where 5 to 25% DMSO and methanol were tested as cryoprotectant, by diluting semen in Ginzburg fish

*gariepinus* is the species native to Africa where it is grown although mostly on a subsistence level for food. The fish is hardy and adaptable to diverse environments even with poor water quality with its air breathing ability (Hecht *et al.,* 1996*). C. gariepinus* is a typically nonaggressive stalking predatory omnivore that hunts at night using non-visual primary sense organs especially the senses of touch through the barbels and tactile organs on the mouth and skin (Bruton, 1996).

The availability of gametes throughout the year is important to ensure a constant supply of fish. In captivity (250C; 12h light per day*), C. gariepinus* gametogenesis is continuous once sexual maturity is reached. However, whereas females can be stripped of eggs after treatment with pituitary extracts, spermatogenesis and male reproductive behavior do not take place spontaneously, even after hormonal therapy. To obtain spermatozoa it is necessary to kill male brood fish or surgically remove the testes. Storing batches of spermatozoa by cryopreservation would significantly improve the reproductive potential of male catfish. The procurement of reliable broodstock (of good genetic quality), fingerlings and as juvenile fish for stocking ponds and fish farms has been a major set back in the development of catfish culture in Nigeria. This is because these cultivable species are not easily obtained from the wild. The development of cryopreservation procedures for sperm of Clarias species will aid in the recovery of threatened and endangered species as well as in the genetic selection and maintenance of lines of selected stocks. Cryopreserved sperm can also benefit commercial aquaculture industry by allowing females to be spawned when males are not available, decreasing the need to hold captive male as broodstock.
