**2.4 Semen collection**

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 syringe.

Cryopreservation of the Sperm

Fig. 1. The African catfish broodstock.

Fig. 2. Male catfish showing the genital openings.

Fig. 3. Removal of testes from male catfish

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 311

#### **2.5 Cryopreservation of sperm, cryoprotective agents used**

Different combination of cryoprotective (CPA) agents are shown in Table 2 as CPA-DP (DMSO+PBS), CPA-DF (DMSO+GFR), CPA-GP (Glycerol+PBS), CPA-GF (Glycerol+GFR), CPA-DGP (DMSO+Glucose+PBS) and CPA-DGF (DMSO+Glucose+GFR). Before cryopreservation of semen, motility of the fresh semen was evaluated in two trials of dilution: 1:1 and 1: 20. In these trials two different extenders were used: Phosphate buffered saline (PBS) and Ginzburg Fish Ringer (GFR). The volume of the extracted semen was measured with a 5.0 ml syringe (DISCARDM(R) NIG ) which was diluted with the extender PBS and GFR for first trial on a ratio 1:1 v/v and 1:10 v/v respectively, then mixed evenly with differently prepared cryoprotective agent combinations at a ratio 1:1 (PBS) for fertility and hatchability evaluation.

In the second trial, the two extenders were used but at different sperm dilution ratio for PBS and GFR at a ratio 1:20 for both extenders. The resulting semen-cryoprotective agent solution in each trial after thorough mixing was dispensed into labeled 1ml cryotubes with a 2-step freezing protocol of first freezing on the chilled water blocks at -100 C for 10 min before it was finally transferred into the liquid nitrogen for a long-term preservation.


Table 1. Composition of the extenders (g/l) tested for cryopreservation of catfish sperm in liquid Nitrogen.


DMSO =Dimethylsulphoxide, PBS =Phosphate buffered saline, GFR=Ginzburg Fish Ringer.: DP = DMSO and PBS; DF = DMSO and GFR; GP = Glycerol + PBS; GF = Glycerol + GFR; DGP = DMSO + PBS + Glucose/Sucrose; DGF = DMSO + GFR + Glucose/Sucrose

Table 2. Composition (%) of the cryoprotective agents used

Fig. 1. The African catfish broodstock.

Different combination of cryoprotective (CPA) agents are shown in Table 2 as CPA-DP (DMSO+PBS), CPA-DF (DMSO+GFR), CPA-GP (Glycerol+PBS), CPA-GF (Glycerol+GFR), CPA-DGP (DMSO+Glucose+PBS) and CPA-DGF (DMSO+Glucose+GFR). Before cryopreservation of semen, motility of the fresh semen was evaluated in two trials of dilution: 1:1 and 1: 20. In these trials two different extenders were used: Phosphate buffered saline (PBS) and Ginzburg Fish Ringer (GFR). The volume of the extracted semen was measured with a 5.0 ml syringe (DISCARDM(R) NIG ) which was diluted with the extender PBS and GFR for first trial on a ratio 1:1 v/v and 1:10 v/v respectively, then mixed evenly with differently prepared cryoprotective agent combinations at a ratio 1:1 (PBS) for fertility

In the second trial, the two extenders were used but at different sperm dilution ratio for PBS and GFR at a ratio 1:20 for both extenders. The resulting semen-cryoprotective agent solution in each trial after thorough mixing was dispensed into labeled 1ml cryotubes with a 2-step freezing protocol of first freezing on the chilled water blocks at -100 C for 10 min

NaCl 8.0 7.0 5.26 KCl 0.02 0.28 0.26 CaCl2 - 0.33 - NaHCO3 0.23 - 0.33 Na2HPO4 1.15 - 0.04 KH2PO4 0.20 - 0.04 Mg SO4 7H2O - - 0.13 C6H12O6 - - 0.66 Table 1. Composition of the extenders (g/l) tested for cryopreservation of catfish sperm in

**agent (CPA) DMSO Glycerol PBS GFR Glucose/** 

DP 10 - 90 - - DF 10 - - 90 - GP - 10 90 - - GF - 10 - 90 - DGP 10 - 85 - 5 DGF 10 - - 85 5

DMSO =Dimethylsulphoxide, PBS =Phosphate buffered saline, GFR=Ginzburg Fish Ringer.: DP = DMSO and PBS; DF = DMSO and GFR; GP = Glycerol + PBS; GF = Glycerol + GFR; DGP = DMSO +

PBS + Glucose/Sucrose; DGF = DMSO + GFR + Glucose/Sucrose Table 2. Composition (%) of the cryoprotective agents used

**Ginzburg Fish Ringer** 

**Calcium-Free HBSS 200mOsmol/kg** 

**Sucrose** 

before it was finally transferred into the liquid nitrogen for a long-term preservation.

**Phosphate buffer saline (PBS)** 

**2.5 Cryopreservation of sperm, cryoprotective agents used** 

and hatchability evaluation.

**Composition (g/1000 ml)** 

liquid Nitrogen.

**Cryoprotective** 

Fig. 2. Male catfish showing the genital openings.

Fig. 3. Removal of testes from male catfish

Cryopreservation of the Sperm

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 313

Fig. 7. Removing the cryopreserved sperm and motility evaluated

Fig. 9. Stripping the eggs from gravid female catfish after injection with Ovaprim

Fig. 8. Rapid thawing process is employed.

Fig. 4. Laceration of testes to extract milt

Fig. 5. Motility evaluation using a microscope and haemacytometer.

Fig. 6. Sperm cryopreserved in liquid Nitrogen for 4-8 months

Fig. 4. Laceration of testes to extract milt

Fig. 5. Motility evaluation using a microscope and haemacytometer.

Fig. 6. Sperm cryopreserved in liquid Nitrogen for 4-8 months

Fig. 7. Removing the cryopreserved sperm and motility evaluated

Fig. 8. Rapid thawing process is employed.

Fig. 9. Stripping the eggs from gravid female catfish after injection with Ovaprim

Cryopreservation of the Sperm

(Hogendoorn, 1979).

**2.7 Method of female stripping** 

as the control for both trials.

**2.9 Motility evaluation** 

follows:

**2.8 Egg clutch variation and fertilization** 

**2.10 Fertility and hatchability evaluation** 

**2.6 Induced spawning and stripping** 

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 315

The readiness of the female broodstock to be used for breeding was tested by holding it in a head-up vertical position and a slight pressure was applied by pressing its abdomen with a thumb from the pectoral fin towards the genital papilla after which eggs run out freely. The selected broodstock were kept separately in different tanks without feeding them, after they were injected with 0.35 ml Ovaprim ® (Syndel, Canada) per kg live weight (Oyeleye and Omitogun, 2007) and then left for 10-12 hours latency period as a post ovulatory maturation period and to ensure high hatching rates and low proportion of deformed larvae

The female body surface was gently dried with clean towel. It was tightly held at both ends by two persons with wet towels and stripped by a gentle press on the abdomen with a thumb towards the rear. The first free running eggs obtained at a slight pressing of the induced female broodstock were collected for fertilization (Legendre and Oteme, 1995).

After inducing, the female fish was stripped and the clutch of eggs weighed (about 150– 160g/kg of the body weight). The eggs were weighed in various measures of 1.0g, 2.0g, 3.0g, 4.0g and 5.0g based on the level of each experiment. That is for 1.0g, it was weighed seven times with replicates for the different cryoprotective agents and control. After which it was fertilized with the cryopreserved semen in liquid nitrogen after thawing in warm water at 350C for 5 min. Fresh semen was used to fertilize same amount of clutches of eggs to serve

The motility of the spermatozoa before and after the addition of the cryopreservative agents, CPA and after thawing was evaluated for each trial. The cryopreserved semen was also further evaluated for fertility, hatchability and survivability for each trial. The motility test was done by diluting a drop of post thawed or fresh spermatozoa either with PBS, GFR or 0.9 % saline solution at a ratio 1:100 from which one drop of the solution was put on the hemocytometer and viewed subsequently under the microscope 10X and 40X, low and high power objectives of the microscope. The result arrived at is converted to the total number of spermatozoa per ml by multiplying it by the dilution factor (100) and 104 (SIGMA, 1994) as

*Total no of spermatozoa per ml = Average No. of counted spermatozoa x 104* 

The development process from fertilized eggs to hatching is dependent upon water temperature while hatching rate is, next to egg quality, dependent on the water quality;

*of the cryopreserved semen* (1)

Fig. 10. Eggs divided into various clutch weights in Petri dishes

Fig. 11. Fertilized eggs incubated in aerated plastic containers covered with nets

Fig. 12. The temperature was kept at 25-27° C and sometimes covered with black plastic sheets.

#### **2.6 Induced spawning and stripping**

314 Current Frontiers in Cryopreservation

Fig. 10. Eggs divided into various clutch weights in Petri dishes

Fig. 11. Fertilized eggs incubated in aerated plastic containers covered with nets

Fig. 12. The temperature was kept at 25-27° C and sometimes covered with black plastic

sheets.

The readiness of the female broodstock to be used for breeding was tested by holding it in a head-up vertical position and a slight pressure was applied by pressing its abdomen with a thumb from the pectoral fin towards the genital papilla after which eggs run out freely. The selected broodstock were kept separately in different tanks without feeding them, after they were injected with 0.35 ml Ovaprim ® (Syndel, Canada) per kg live weight (Oyeleye and Omitogun, 2007) and then left for 10-12 hours latency period as a post ovulatory maturation period and to ensure high hatching rates and low proportion of deformed larvae (Hogendoorn, 1979).

#### **2.7 Method of female stripping**

The female body surface was gently dried with clean towel. It was tightly held at both ends by two persons with wet towels and stripped by a gentle press on the abdomen with a thumb towards the rear. The first free running eggs obtained at a slight pressing of the induced female broodstock were collected for fertilization (Legendre and Oteme, 1995).

#### **2.8 Egg clutch variation and fertilization**

After inducing, the female fish was stripped and the clutch of eggs weighed (about 150– 160g/kg of the body weight). The eggs were weighed in various measures of 1.0g, 2.0g, 3.0g, 4.0g and 5.0g based on the level of each experiment. That is for 1.0g, it was weighed seven times with replicates for the different cryoprotective agents and control. After which it was fertilized with the cryopreserved semen in liquid nitrogen after thawing in warm water at 350C for 5 min. Fresh semen was used to fertilize same amount of clutches of eggs to serve as the control for both trials.

#### **2.9 Motility evaluation**

The motility of the spermatozoa before and after the addition of the cryopreservative agents, CPA and after thawing was evaluated for each trial. The cryopreserved semen was also further evaluated for fertility, hatchability and survivability for each trial. The motility test was done by diluting a drop of post thawed or fresh spermatozoa either with PBS, GFR or 0.9 % saline solution at a ratio 1:100 from which one drop of the solution was put on the hemocytometer and viewed subsequently under the microscope 10X and 40X, low and high power objectives of the microscope. The result arrived at is converted to the total number of spermatozoa per ml by multiplying it by the dilution factor (100) and 104 (SIGMA, 1994) as follows:

*Total no of spermatozoa per ml = Average No. of counted spermatozoa x 104* 

*of the cryopreserved semen* (1)

#### **2.10 Fertility and hatchability evaluation**

The development process from fertilized eggs to hatching is dependent upon water temperature while hatching rate is, next to egg quality, dependent on the water quality;

Cryopreservation of the Sperm

**and egg clutch fertilization** 

and 1: 40 and for the control.

The various extenders used were 200mOsmol/kg Ca-F HBSS 300 mOsmol/kg Ca-F HBSS 400 mOsmol/kg Ca-F HBSS

replicates were made for each treatment.

**2.12 The control** 

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 317

The control for both trials was prepared by the use of fresh semen obtained from the lacerated testes from a normal gravid male broodstock with the use of sterilized dissecting knives but activated with saline solution in the ratio of 1:1 v/v and subsequently used to fertilized various measures of egg clutches ranging from 1.0g, 2.0g, 3.0g, 4.0g and 5.0g normal eggs from the same batch of eggs i.e. from the same fish. Control was set up for the evaluation of each parameter for trials *i.e.,* motility, fertility, hatchability and survivability. The motility evaluation of the post-thawed cryopreserved sperm was evaluated after

dilution with the extender on a ratio 1:100 using hemocytometer (SIGMA, 1994).

for the next seven months in liquid Nitrogen stored in Dewar container

before the final transfer into liquid nitrogen for the next 4 to 7 months.

**2.14 Experiment on storing sperm in refrigerator** 

RPMI 1640 (SIGMA) Culture Medium in 0.9% NaCl Solution

**2.13 Further scaling up for commercial application of cryopreserved semen dilution** 

Motility of the fresh semen was evaluated in two different trials of dilution, before any cryopreservation of the semen. Phosphate-buffered-saline (PBS) and ordinary saline water were the two extenders used in the two different trials and were diluted 1:1 and 1:40. The extracted semen volume was measured with a 5.0ml syringe (DISCARDIM(R)NIG) which was diluted with the extender PBS for trial on a ratio 1:20v/v and 1:200v/v respectively and thereafter mixed evenly with the cryoprotective agents at a ratio of 1:1 and cryopreserved

For the second trials, the same cryoprotectant (85% PBS+5% glucose+10% DMSO) was used but at different sperm dilution ratio of 1:40. In both trials, the resulting semen in each experiment after thorough mixing was then dispensed into labeled 1ml cryotubes while a 2 step freezing protocol, e.g. initial freezing onto frozen water (ice) blocks at -100C for 30min

After induction with Ovaprim, the female broodstock was stripped and the clutch of eggs weighed (which was about 150-200g/kg body weight) and divided into three portions of about 120 g each for the experiment on fertilization with cryopreserved sperm of diluted 1:1

This procedure was repeated three times in a nearby commercial farm 5kms away from the University to serve as the replicates and to ensure the repeatability of the experiments.

The three different osmolalities of extender Ca-FHBSS (Calcium-free Hanks Balanced Salt Solution) were prepared according to Riley, 2002. The sperm with the 200, 300 and 400 mOsmol/kg Ca-F HBSS were kept in the refrigerator at 40C. The semen samples with RPMI and 0.9% NaCl solutions were kept at both room temperature and refrigerator. Two

temperature, oxygen level, pH and water hardness. After stripping of the induced female broodstock, the eggs were weighed in grams depending on the on-going experiment *i.e*, 1.0g (600+100 eggs), 2.0g, etc. The various measures of eggs (repeated 12 times together with replicates) were fertilized with cryopreserved semen thawed at 350C for 5 minutes, and a pair of egg clutches with fresh semen as control experiment. The mixture of eggs and semen was stirred gently for at least 1.0 min to allow contact and adequate fertilization. Within a few minutes after fertilization the eggs absorbed water and could become sticky so the eggs were distributed in a netted basket suspended in the hatching trough (50cm x 35cm x 30cm) containing contaminant-free (passed through a purification system with ultraviolet sterilization at 3000 μW/cm2) well-aerated water in a single layer so that the eggs get sufficient oxygen during incubation. The hatching troughs were completely covered with mosquito net and black polythene materials placed under 200 Watt bulbs to prevent mosquitoes and other insects laying eggs and to increase level of heat generation. The system was supplied with an electric aerator to increase level of oxygen dissolved in the water.

The incubated eggs were monitored and temperature maintained between 260C -270C for incubation between 23– 25 h. Soon after hatching the larvae passed through the net and the dead eggs and shells remain on the net in the basket. The larvae were then simply separated from the unfertilized eggs and eggshells by lifting the basket and the nets out of the hatching trough.

The percentage, % fertility and hatchability were determined subjectively after 12–15 h of fertilization by identifying the healthy developing eggs which were transparent green brownish in colour (Coppens, 2007) while the dead eggs were also estimated:

$$\text{\%} \text{ \textquotedbl{} Vertillity =} \text{(No. of \text{\textquotedbl{}}ctrlilized \text{ eygs} / No. of \text{\textquotedbl{}} insemimated \text{egs}) X 100\text{\textquotedbl{}} \text{\textquotedbl{}}} \tag{2}$$

$$\% \text{ Attackability} = \frac{\text{Total No. of ferrtillized eggs} - \text{No. of unhatched edges}}{\text{Total No. of ferrtillized eggs}} \times 100\% \tag{3}$$

#### **2.11 Post-hatching survivability evaluation**

This is done by allowing the newly hatched larvae of all the treatments and that of the control to live on the remains of their yolk sacs for the first 4 days (Heicht *et al*.,1996) after hatching out of the eggs and thereafter carefully removed from the hatching troughs and were fed with Artemia (Inve Aquaculture, USA) on a regular basis (*i.e.,* twice per day). Irregularities in the activities of the fry in terms of feeding, movement in water was observed at the same time taking note of the dead fry which were removed immediately to avoid contamination of water. Survivability evaluation which was observed for a period of about 3 – 4 weeks was done for each stage of the experiment together with fertility and hatchability for fresh (control experiment) and cryopreserved spermatozoa. The posthatching survivability was evaluated as follows:

$$\% \text{ Surivability} = \frac{\text{Total No. of } \text{ larvaae} \quad \text{ - No. of } \text{ dead larvaae}}{\text{Total No. of } \text{ larvaae}} \times 100\% \tag{4}$$

#### **2.12 The control**

316 Current Frontiers in Cryopreservation

temperature, oxygen level, pH and water hardness. After stripping of the induced female broodstock, the eggs were weighed in grams depending on the on-going experiment *i.e*, 1.0g (600+100 eggs), 2.0g, etc. The various measures of eggs (repeated 12 times together with replicates) were fertilized with cryopreserved semen thawed at 350C for 5 minutes, and a pair of egg clutches with fresh semen as control experiment. The mixture of eggs and semen was stirred gently for at least 1.0 min to allow contact and adequate fertilization. Within a few minutes after fertilization the eggs absorbed water and could become sticky so the eggs were distributed in a netted basket suspended in the hatching trough (50cm x 35cm x 30cm) containing contaminant-free (passed through a purification system with ultraviolet sterilization at 3000 μW/cm2) well-aerated water in a single layer so that the eggs get sufficient oxygen during incubation. The hatching troughs were completely covered with mosquito net and black polythene materials placed under 200 Watt bulbs to prevent mosquitoes and other insects laying eggs and to increase level of heat generation. The system was supplied with an electric aerator to increase level of oxygen dissolved in the

The incubated eggs were monitored and temperature maintained between 260C -270C for incubation between 23– 25 h. Soon after hatching the larvae passed through the net and the dead eggs and shells remain on the net in the basket. The larvae were then simply separated from the unfertilized eggs and eggshells by lifting the basket and the nets out of the

The percentage, % fertility and hatchability were determined subjectively after 12–15 h of fertilization by identifying the healthy developing eggs which were transparent green

*% Fertility =(No. of fertilized eggs / No. of inseminated egg) X 100%* (2)

. – . % 100% .

This is done by allowing the newly hatched larvae of all the treatments and that of the control to live on the remains of their yolk sacs for the first 4 days (Heicht *et al*.,1996) after hatching out of the eggs and thereafter carefully removed from the hatching troughs and were fed with Artemia (Inve Aquaculture, USA) on a regular basis (*i.e.,* twice per day). Irregularities in the activities of the fry in terms of feeding, movement in water was observed at the same time taking note of the dead fry which were removed immediately to avoid contamination of water. Survivability evaluation which was observed for a period of about 3 – 4 weeks was done for each stage of the experiment together with fertility and hatchability for fresh (control experiment) and cryopreserved spermatozoa. The post-

. – . % 100% .

*Total No of larvae No of dead larvae Survivability Total No of larvae* (4)

*Total No of fertilized eggs No of unhatcched eggs Hatchability Total No of Fertilized eggs* (3)

brownish in colour (Coppens, 2007) while the dead eggs were also estimated:

**2.11 Post-hatching survivability evaluation** 

hatching survivability was evaluated as follows:

water.

hatching trough.

The control for both trials was prepared by the use of fresh semen obtained from the lacerated testes from a normal gravid male broodstock with the use of sterilized dissecting knives but activated with saline solution in the ratio of 1:1 v/v and subsequently used to fertilized various measures of egg clutches ranging from 1.0g, 2.0g, 3.0g, 4.0g and 5.0g normal eggs from the same batch of eggs i.e. from the same fish. Control was set up for the evaluation of each parameter for trials *i.e.,* motility, fertility, hatchability and survivability.

The motility evaluation of the post-thawed cryopreserved sperm was evaluated after dilution with the extender on a ratio 1:100 using hemocytometer (SIGMA, 1994).

#### **2.13 Further scaling up for commercial application of cryopreserved semen dilution and egg clutch fertilization**

Motility of the fresh semen was evaluated in two different trials of dilution, before any cryopreservation of the semen. Phosphate-buffered-saline (PBS) and ordinary saline water were the two extenders used in the two different trials and were diluted 1:1 and 1:40. The extracted semen volume was measured with a 5.0ml syringe (DISCARDIM(R)NIG) which was diluted with the extender PBS for trial on a ratio 1:20v/v and 1:200v/v respectively and thereafter mixed evenly with the cryoprotective agents at a ratio of 1:1 and cryopreserved for the next seven months in liquid Nitrogen stored in Dewar container

For the second trials, the same cryoprotectant (85% PBS+5% glucose+10% DMSO) was used but at different sperm dilution ratio of 1:40. In both trials, the resulting semen in each experiment after thorough mixing was then dispensed into labeled 1ml cryotubes while a 2 step freezing protocol, e.g. initial freezing onto frozen water (ice) blocks at -100C for 30min before the final transfer into liquid nitrogen for the next 4 to 7 months.

After induction with Ovaprim, the female broodstock was stripped and the clutch of eggs weighed (which was about 150-200g/kg body weight) and divided into three portions of about 120 g each for the experiment on fertilization with cryopreserved sperm of diluted 1:1 and 1: 40 and for the control.

This procedure was repeated three times in a nearby commercial farm 5kms away from the University to serve as the replicates and to ensure the repeatability of the experiments.

#### **2.14 Experiment on storing sperm in refrigerator**

The various extenders used were


The three different osmolalities of extender Ca-FHBSS (Calcium-free Hanks Balanced Salt Solution) were prepared according to Riley, 2002. The sperm with the 200, 300 and 400 mOsmol/kg Ca-F HBSS were kept in the refrigerator at 40C. The semen samples with RPMI and 0.9% NaCl solutions were kept at both room temperature and refrigerator. Two replicates were made for each treatment.

Cryopreservation of the Sperm

for egg weights 3.0g and 4.0g.

**and fertility** 

trial.

lowest result.

extenders.

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 319

decreasing order of C > DGP > DP > GP > GF. Generally, DGP gave the best followed by DP (without 5% glucose) while Glycerol in combination with Ginsburg fish ringer gave the

A close means values were also discovered with trial 1 which indicate the results were better in trial 2, the further diluted semen which could supposedly be explained by addition of

The differences in fertility and hatchability with control and cryoprotectants tested may be due to the mild damage done to the spermatozoa during the process of lacerating the testes to extract the semen, and also due to the intracellular vitrification (Cryobiosystems, 2009)- a

There was a significant effect (p**<**0.05) of egg clutch weight on fertility, hatchability and survivability. In trial 1, although, the hatchability increases with increase in egg weight but the increment at egg weight 4.0g and 5.0g was not significantly different (LSD=88.749, p>0.05). There was fertility optimization at 4.0 g of egg clutch weight which though, close to the mean values of 5.0g which is higher but not a uniform increase. The same trend was observed for fertility of eggs but much higher than corresponding hatchability which may be due to loss of eggs to external factors like temperature, contamination and possible error during record taking. There is no significant difference for fertility at egg clutch weight 3.0g and 4.0g but there was significant difference (p<0.05) from 5.0g. Survivability was not

In the second trial, a similar trend was observed; hatchability was highest (p<0.05) at egg clutch weight 4.0g. No significant difference (p>0.05) in survivability was observed except

Generally, for both trials, egg clutch weight at 4.0 g gave the optimum viability value.

cryoprotective agent (CPA) and egg clutch weight on fertility and hatchability.

**3.3 Effect of type of cryoprotectant and egg clutch weight interaction on hatchability** 

From the statistical analysis, it showed that there could also be effect of interaction of both

The result, as observed shows a significant effect of (p<0.05) of interaction of CPA and egg clutch weight on fertility, hatchability for the first trial and only on fertility for the second

Effect of interaction of cryoprotectant on hatchability was not different from the trend of results obtained in previous results. However, the cryoprotective agents were not significantly different (p>0.05) from each other but DGP, GP and DP still maintained the higher mean values while control took the highest. DP and DGP were not significantly different (p>0.05) from each other for egg clutch weight such as 1.0g to 5.0g. However, GP was significantly different (P<0.05) from DGP and DP for egg clutch weight 2.0-5.0g. For GF, DF and DGF, there was also no significant effect (p>0.05) with changes in egg weights. The effect of interaction of both CPA and egg weights on fertility was also significant (p<0.05).

commonly occurring problem in the process of cryopreservation in liquid Nitrogen.

**3.2 Effects of egg clutch weight on viability of African catfish gametes** 

significantly different from each other except for 5.0g (p<0.05).
