**1.1 Success of cryopreservation in African catfish in Nigeria**

Cryopreservation of fish spermatozoa has been the subject of many investigations. Successful cryopreservation depends not only on the right choice of cryoprotectant and extender, but also on the freezing protocol used. Cryoprotectant and freezing rate together determine the damage to spermatozoa due to intracellular ice crystallization.

The first two years of the NACGRAB- OAU Department of Animal Sciences cryopreservation project (2005-2007) were dedicated to optimization of cryopreservation protocols of the catfish sperm under short-term condition in deep freezer at -10 to -30°C (Oyeleye and Omitogun, 2007) and testing the viability of cryopreserved sperm by studying the ability of these cryopreserved sperm in fertilizing freshly spawned eggs (Omitogun *et al.*, 2006).

The second phase of the project (2008-2010) was dedicated to cryopreservation of the catfish sperm under long term conditions in liquid Nitrogen (-2900C) and testing the motility and ability to fertilize eggs. Evaluation of optimization and economic feasibility of cryopreserved sperm was also carried out (Omitogun *et al.*, 2010). To this end, cryopreserved sperm in liquid nitrogen in Dewar container was further diluted and cryopreserved from 3-8 months, then was taken to an identified and willing commercial catfish farm with the objective of testing the ability of cryopreserved sperm of African giant catfish to fertilize a whole clutch of eggs from a mature female catfish, normally used by commercial farmers and consequently confirm the viability of using cryopreserved sperm in normal commercial hatchery operations. Our hypothesis was that if cryopreseved sperm is practically tested on

*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

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.

determine the damage to spermatozoa due to intracellular ice crystallization.

Cryopreservation of fish spermatozoa has been the subject of many investigations. Successful cryopreservation depends not only on the right choice of cryoprotectant and extender, but also on the freezing protocol used. Cryoprotectant and freezing rate together

The first two years of the NACGRAB- OAU Department of Animal Sciences cryopreservation project (2005-2007) were dedicated to optimization of cryopreservation protocols of the catfish sperm under short-term condition in deep freezer at -10 to -30°C (Oyeleye and Omitogun, 2007) and testing the viability of cryopreserved sperm by studying the ability of these cryopreserved sperm in fertilizing freshly spawned eggs (Omitogun *et al.*,

The second phase of the project (2008-2010) was dedicated to cryopreservation of the catfish sperm under long term conditions in liquid Nitrogen (-2900C) and testing the motility and ability to fertilize eggs. Evaluation of optimization and economic feasibility of cryopreserved sperm was also carried out (Omitogun *et al.*, 2010). To this end, cryopreserved sperm in liquid nitrogen in Dewar container was further diluted and cryopreserved from 3-8 months, then was taken to an identified and willing commercial catfish farm with the objective of testing the ability of cryopreserved sperm of African giant catfish to fertilize a whole clutch of eggs from a mature female catfish, normally used by commercial farmers and consequently confirm the viability of using cryopreserved sperm in normal commercial hatchery operations. Our hypothesis was that if cryopreseved sperm is practically tested on

**1.1 Success of cryopreservation in African catfish in Nigeria** 

and skin (Bruton, 1996).

2006).

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 in the dry season.

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

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

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

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

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

Cryopreservation of the Sperm

of the tank for easy flow by gravity.

**2.2 Selection of broodstock** 

maintained at 25–270C.

**2.4 Semen collection** 

syringe.

**2.3 Preparation of extender-cryoprotectant** 

avoid contamination and deterioration of the spermatozoa.

regulated through proper monitoring and replaced weekly.

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 309

The matured male and female broodstock were kept at constant temperature of 270C in a 1000 litre tank connected to a source of water by a pipe connected from the reservoir plastic tank placed in an elevated stand in the laboratory and its drainage was located at the bottom

The broodstock were fed with an imported floating palletized feed *i.e., CoppensR* feed (42% protein, ISO -170 certified, Netherlands) containing a large percentage of high quality fish meal, which is especially important to facilitate repeated spawning at a maintenance level of 1.5% body weight on a daily basis by gradual hand broadcast. The water quality was

The sexually matured female was selected according to their swollen, reddish genital papilla and a well distended, swollen soft abdomen. A slight pressure was applied on the abdomen towards the genital papilla after which ripe eggs oozed out which were green-brownish in color and ripe eggs are generally uniform in size. The female broodstock was stocked in the hatchery for about 2 days without feeding so that the alimentary tract was empty at the time of stripping. It is very important that the collected eggs did not get contaminated. Sexually matured male broodstock was selected based on a reddish or pinkish pointed and vascularized genital papilla. The temperature of the broodstock kept in the tank was

Two extenders were used in this study, phosphate buffered saline (PBS) and Ginzburg Fish Ringer (GFR) with pH of 7.4 and 7.6 respectively. The extenders were prepared as shown in Table 1 with Calcium-free Hanks Balanced Salt Solution (Ca-FHBSS) used in experiment 2. 14; after which they were sterilized for 20 min at 15 lbs/inch2 using a pressure cooker to

A good quantity of the sperm of African catfish cannot be stripped and sperm can only be obtained after sacrificing it. Sexually matured male weighing 0.8+ 0.2kg were selected and kept in a different tank of about 50 l capacity for about 18 h prior to the time of sperm collection .The male broodstock was dried with clean towel and then made unconscious by breaking its backbone. The body cavity was carefully opened with a pair of sterilized scissors without damaging the testes after which the two testes were dissected out. It was then removed with a pair of forceps, the blood veins cleared out and rinsed in saline solution. The testes were lacerated with a new and sharp razor blade; the milt was gently squeezed out and collected in a sterilized Petri dish. The whole process was carried out in a disinfected environment to avoid bacterial contamination which can lead to degradation of samples, transfer of pathogens and inaccurate estimation of motility. Sterilized instrument and aseptic techniques for collection of sperm was incorporated to reduce the contamination by bacteria. The volume of the extracted sperm was measured with a 5.0 ml sterilized

ringer and freezing in 1-milliliter cryovials in a programmable freezer. To avoid an excess of spermatozoa per egg, post-thaw semen was diluted 1:20, 1:200 or 1:2000 before fertilization. Even frozen- thawed spermatozoa with low numbers of live cells yielded adequate hatching rates. They found out that the maximum sperm dilution ratio to achieve hatching rates similar to control was 1:200 without losing fertilization ability. However, at 1:2000 the hatching rates produced with frozen spermatozoa were lower than the control African catfish. Similarly *Heterobranchus longifilis* spermatozoa were diluted 1:3 before freezing and 1:10 after thawing and had the same fertilization ability (78.9%) as the control (81.1%). On the contrary for *Cyprinus carpio*, no spermatozoa survived when diluted higher than 1: 5 before and after freezing (Lubzens *et al.*, 1997).

Cryopreservation of catfish spermatozoa is useful as a routine method of gamete storage and management. However, the economic factor should also be considered. The technology of cryopreservation with the use of liquid nitrogen though desirable but is cost intensive. Therefore there is a need to study how the cryopreserved semen will be maximally utilized with good fertilizing results at the same time cost-effective and affordable for the farmers

To avoid wastage of cryopreserved spermatozoa per egg clutch after dilution with physiological salt solution, fertilization of various measures of egg clutches were tested in the present research with differently cryopreserved spermatozoa for optimization and for cost evaluation. In a second study the concentration of the semen was reduced, *i.e*., diluted at a dilution ratio of 1:20 and 1:200 to verify the spermatozoa are not in excess and consequently be wasted.

Another study was carried out in order to assist the farmers to determine the approximate amount of egg clutch that will be adequate for a milliliter (ml) of cryopreserved semen without wasting spermatozoa in order to evaluate economic cost and profitability. The aim of this study was to verify the possibility of cryopreserving African catfish under long-term condition in liquid Nitrogen (LN2) and evaluate the viability and fertility optimization of a specific amount (*i.e.,* 1 ml ) of cryopreserved semen of African catfish cryopreserved in LN2 using various cryoprotective agents with different measures of egg clutches. This paper aimed to establish a standard fertility ratio between a ml of semen and clutch of eggs in order to prevent wastage of semen; be able to maximize the resources and evaluate profitability of cryopreservation in liquid nitrogen by evaluating the effects on the cryopreserved semen as to motility and hatchability, the ability to hatch the eggs from a gravid female and survival of ensuing larvae.
