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

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

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 for egg weights 3.0g and 4.0g.

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

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

From the statistical analysis, it showed that there could also be effect of interaction of both cryoprotective agent (CPA) and egg clutch weight on fertility and hatchability.

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 trial.

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).

Cryopreservation of the Sperm

semen (50%) (P>0.05).

which in turn was favourable for survival.

fresh semen

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 321

Fig. 13. Motilities of cryopreserved semen at ratios (1:1, and 1:40) compared with the control

Cryopreservation in liquid Nitrogen did not have any effect on the survival of *C. gariepinus* larvae produced from cryopreserved semen as shown. However, larvae produced by the cryopreserved semen gave a higher survival, ratio 1:1 (65%), Ratio 1:40 (63%) and fresh

The present study proves that sperm of African catfish cryopreserved aged up to 7 months in liquid Nitrogen and diluted more than 40 times with the extender is viable. The reason for low survivability rate in the control experiment using fresh semen may be attributed to high stocking density (because of the greater number of surviving fry) as practiced by many farmers, *i.e* the quantity of larvae per unit volume of water is less for cryopreserved sperm

This experiment evaluated the effect of extenders and period of refrigerated storage on the sperm motility of *Clarias gariepinus* sperm cells with the intent to identify a suitable extender for the refrigerated storage of the sperm cells of *Clarias gariepinus.* Semen samples were collected from mature broodstock and were refrigerated with various different extenders at ratio 1:3 namely: Calcium-free Hanks' Balanced Salt Solution (Ca-F HBSS), RPMI 1640 culture medium and 0.9% NaCl. Ca-F HBSS extender was prepared at 3 different osmolalities: 200mOsmol/kg, 300mOsmol/kg and 400mOsmol/kg. Sperm in RPMI 1640 and 0.9% NaCl extenders were also kept at room temperature to assess the effect of refrigeration on motility of catfish sperm cells. Motility was monitored on a 24-hour basis and % motility was evaluated daily. Results showed that sperm cells of *Clarias gariepinus*  using 200mOsmol/kg as extender (p<0.05) can be stored under refrigeration for 12 days. However, of all the extenders evaluated, RPMI 1640 proved to be the most effective extender

**3.5 Motility evaluation of refrigerated catfish sperm cells in different extenders** 

(p<0.05) retaining higher motility of the refrigerated sperm cells of *Clarias gariepinus.* 

The semen samples extended with 0.9% NaCl and the RPMI culture solution at room temperature did not have motile sperm cells after 48 hours. The motility of the semen

**3.5.1 Effect of refrigeration on % motility of sperm cells** 

**1:01 1:40 fresh**

The results for both trials were also very close, following the same trend, except for control changes in egg clutch weights from 1.0g to 5.0g was generally not significantly different for DP and DGP followed by GP and GF which were not significantly different (p>0.05) from means values of egg clutch weight 4.0g–5.0g for DGP and DP. However, GF gave the lowest value for the two trials.

#### **3.4 Scaling up of the applications of the cryopreserved semen for commercial aquaculture**

#### **3.4.1 Effect of dilution ratio on viability of catfish gametes**

Table 3 shows the comparison for the parameters measured among the control, dilution ratios 1:1 and 1:40. The fresh semen gave the highest fertility and hatchability rates (P<0.05). It is significantly different from ratio 1:1 and ratio 1:40. Comparing the fertility and hatchability rates of the two different dilution rates, ratio 1:1 gave the highest fertility and hatchability rates which was significant (P<0.05). Survival rate however, followed a different trend in which dilution ratio 1:1 gave the highest survival rate closely followed by ratio 1:40 while the control semen gave the least survival rate (P< 0.05).

Most importantly, the differences in fertility and hatchability may also be attributed to the condition of the farmer's hatchery environment in which many environmental and sanitation conditions were compromised for maximum profit (Amupitan *et al.,* 2010).


Means in the same column with different letter are significantly different at P<0.05, C=Control, LSD = Least Significant Difference

Table 3. Effects of dilution ratio on viability of Catfish semen diluted at ratio 1:1 and ratio 1:40

#### **3.4.2 Effect of dilution ratio on motility of catfish semen and survival of ensuing larvae**

The fresh semen gave the highest motility at appreciably high percentage (71.00%) which was significantly different (P<0.05) from cryopreserved semen diluted at ratios 1:1 (50.52%) and 1:40 (49.05%) (Fig. 13). However, there was no significant difference between the two diluted cryopreserved semen (P>0.05). It was evident that the freezing process and cryopreservation decreased sperm motility after cryopreservation. It could be deduced that cryopreserved sperm still needs to be completely activated after thawing in order to fertilize the whole clutch of eggs since there is a direct relationship between motility and fertility.

The results for both trials were also very close, following the same trend, except for control changes in egg clutch weights from 1.0g to 5.0g was generally not significantly different for DP and DGP followed by GP and GF which were not significantly different (p>0.05) from means values of egg clutch weight 4.0g–5.0g for DGP and DP. However, GF gave the lowest

Table 3 shows the comparison for the parameters measured among the control, dilution ratios 1:1 and 1:40. The fresh semen gave the highest fertility and hatchability rates (P<0.05). It is significantly different from ratio 1:1 and ratio 1:40. Comparing the fertility and hatchability rates of the two different dilution rates, ratio 1:1 gave the highest fertility and hatchability rates which was significant (P<0.05). Survival rate however, followed a different trend in which dilution ratio 1:1 gave the highest survival rate closely followed by ratio 1:40

Most importantly, the differences in fertility and hatchability may also be attributed to the condition of the farmer's hatchery environment in which many environmental and

**RATIO MOTILITY FERTILITY HATCHABILITY SURVIVAL**  1:1 55%b 30% b 35%b 15%a 1:40 49%b 29%c 34%c 14%b C 72%a 54%a 62%a 13%c LSD 13% 9.2% 25% 13%

sanitation conditions were compromised for maximum profit (Amupitan *et al.,* 2010).

Means in the same column with different letter are significantly different at P<0.05, C=Control,

**3.4.2 Effect of dilution ratio on motility of catfish semen and survival of ensuing** 

Table 3. Effects of dilution ratio on viability of Catfish semen diluted at ratio 1:1 and ratio

The fresh semen gave the highest motility at appreciably high percentage (71.00%) which was significantly different (P<0.05) from cryopreserved semen diluted at ratios 1:1 (50.52%) and 1:40 (49.05%) (Fig. 13). However, there was no significant difference between the two diluted cryopreserved semen (P>0.05). It was evident that the freezing process and cryopreservation decreased sperm motility after cryopreservation. It could be deduced that cryopreserved sperm still needs to be completely activated after thawing in order to fertilize the whole clutch of eggs since there is a direct relationship between motility and

**3.4 Scaling up of the applications of the cryopreserved semen for commercial** 

**3.4.1 Effect of dilution ratio on viability of catfish gametes** 

while the control semen gave the least survival rate (P< 0.05).

value for the two trials.

**aquaculture** 

**DILUTION** 

1:40

**larvae** 

fertility.

LSD = Least Significant Difference

Fig. 13. Motilities of cryopreserved semen at ratios (1:1, and 1:40) compared with the control fresh semen

Cryopreservation in liquid Nitrogen did not have any effect on the survival of *C. gariepinus* larvae produced from cryopreserved semen as shown. However, larvae produced by the cryopreserved semen gave a higher survival, ratio 1:1 (65%), Ratio 1:40 (63%) and fresh semen (50%) (P>0.05).

The present study proves that sperm of African catfish cryopreserved aged up to 7 months in liquid Nitrogen and diluted more than 40 times with the extender is viable. The reason for low survivability rate in the control experiment using fresh semen may be attributed to high stocking density (because of the greater number of surviving fry) as practiced by many farmers, *i.e* the quantity of larvae per unit volume of water is less for cryopreserved sperm which in turn was favourable for survival.

#### **3.5 Motility evaluation of refrigerated catfish sperm cells in different extenders**

This experiment evaluated the effect of extenders and period of refrigerated storage on the sperm motility of *Clarias gariepinus* sperm cells with the intent to identify a suitable extender for the refrigerated storage of the sperm cells of *Clarias gariepinus.* Semen samples were collected from mature broodstock and were refrigerated with various different extenders at ratio 1:3 namely: Calcium-free Hanks' Balanced Salt Solution (Ca-F HBSS), RPMI 1640 culture medium and 0.9% NaCl. Ca-F HBSS extender was prepared at 3 different osmolalities: 200mOsmol/kg, 300mOsmol/kg and 400mOsmol/kg. Sperm in RPMI 1640 and 0.9% NaCl extenders were also kept at room temperature to assess the effect of refrigeration on motility of catfish sperm cells. Motility was monitored on a 24-hour basis and % motility was evaluated daily. Results showed that sperm cells of *Clarias gariepinus*  using 200mOsmol/kg as extender (p<0.05) can be stored under refrigeration for 12 days. However, of all the extenders evaluated, RPMI 1640 proved to be the most effective extender (p<0.05) retaining higher motility of the refrigerated sperm cells of *Clarias gariepinus.* 

#### **3.5.1 Effect of refrigeration on % motility of sperm cells**

The semen samples extended with 0.9% NaCl and the RPMI culture solution at room temperature did not have motile sperm cells after 48 hours. The motility of the semen

Cryopreservation of the Sperm

solution in which they are suspended in.

samples extended at room temperature.

at 24 h being 34.46% (Fig. 15).

16.93%, 10.73%, 2.77% and 2.28%).

*gariepinus*.

**3.5.2 Effect of osmolality of Ca-F HBSS on % motility** 

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 323

The art of refrigeration provides a low temperature which lowers the metabolic rate of living organisms. The extended semen samples maintained at room temperature proceeded at the normal metabolic rate, hence the sperm cells could not survive up to 48 h. When the sperm cells were still within the fish in the testis, they were supplied with nutrients and the waste they produced are excreted out of the testis, they cannot be supplied with energy or nutrients except provided externally as in tissue culture. Their wastes also accumulate in the

High metabolic rate means faster rate of using up available resources by living organism such as nutrients and energy. It also means that waste will be produced at a faster rate thereby causing fast accumulation of waste in the solution in which the sperm cells are suspended. This accumulation will immediately reach a toxic level causing fatality in the sperm cells. Whereas the low temperature provided by the refrigerator to the refrigerated semen sample reduced the metabolic rate of the sperm cells, thereby reducing the rate at which the available nutrients and energy in the semen-extender solution are used up. The nutrient and energy in the semen-extender solution lasted a much longer period when refrigerated, thus keeping the sperm cells alive for a longer period than in the semen

After 24 hours, the motility of the refrigerated semen sample with the 200mOsmol/kg Ca-F HBSS dropped to 34.85% and motility was retained till the 12thday (288 hours) with 0.5% motility. The refrigerated semen sample with 300mOsmol/kg Ca-F HBSS retained motility for 10 days with % motility at 24 h being 31.88 and motility by the tenth day had dropped to 2.28%. The refrigerated semen sample with 400mOsmol/kg Ca-F HBSS retained motility also for 10 days but with a lower % motility at the 10th day (0.4% motility) but with motility

Based on the length of days for which motility was retained, the 200mOsmol/kg Ca-F HBSS proved to be a good extender since it retained motility for 12 days but with a very low motility (0.5%) However the 300mOsmol/kg, although retained motility for only 10 days, is better since it had the highest motility at the fourth, seventh, ninth and tenth day (i.e.

A good extender should be isotonic to the seminal plasma of the fish. This is to keep the sperm cells immotile until ready for use. Sperm cells are immotile in the seminal plasma and when semen is released in aquatic environment, osmolality goes down and motility is initiated in freshwater species. (Maria *et al.*, 2006). Motility in freshwater species is initiated by exposure of the semen to a hypotonic solution (Morisawa and Suzuki, 1980). Use of extender solutions that are similar in chemical concentration and osmolality are essential to optimizing storage time (Baynes *et al.*, 1981). According to Mansour *et al.,* 2002, motility of *Clarias gariepinus* is completely but irreversibly suppressed in electrolytes and non-electrolytes with an osmolality of 200mOsmol/kg. This statement by Mansour *et al.,*  2002 proves that the osmolality of the seminal plasma of *Clarias gariepinus* is less than or equal to 200mOsmol/kg. This explains why the 200mOsmol/kg of Ca-F HBSS retained motility till the twelfth day as it is closer to being isotonic to the seminal plasma of *Clarias* 

sample with 0.9% NaCl at room temperature dropped from the initial motility of 74.82% to 6.24% (Fig.3.6. 1) after the first 24 h while the semen sample with the RPMI culture solution at room temperature had 0.4% motility at the end of 24 h. The semen sample with 0.9% NaCl at room temperature had a fishy irritating smell after 24 h. This may be due to the production of waste since the sperm cells metabolised at the normal rate.

The semen samples with 0.9% NaCl and RPMI 1640 culture solution retained motility much longer when refrigerated (Fig. 14). The refrigerated semen sample with 0.9% NaCl retained motility for up to 7 days with motility after 24, 48, 72, and 168 hourly being 34.75%, 17.03%, 14.06% and 4.46% respectively. The refrigerated semen sample with RPMI however kept for 9 days with motility after 24, 48, 72, 168 and 216 hours being 53.47%, 37.62%, 25.64%, 8.32% and 5.25% respectively.

Fig. 14. Effect of refrigeration on motility of semen sample extended with NaCl (left) and RPMI (right). The refrigerated semen sample retained motility till the 7th (in NaCl) and 9th day (in RPMI) whereas semen samples at room temperature only lasted till the first day after storage.

sample with 0.9% NaCl at room temperature dropped from the initial motility of 74.82% to 6.24% (Fig.3.6. 1) after the first 24 h while the semen sample with the RPMI culture solution at room temperature had 0.4% motility at the end of 24 h. The semen sample with 0.9% NaCl at room temperature had a fishy irritating smell after 24 h. This may be due to the

The semen samples with 0.9% NaCl and RPMI 1640 culture solution retained motility much longer when refrigerated (Fig. 14). The refrigerated semen sample with 0.9% NaCl retained motility for up to 7 days with motility after 24, 48, 72, and 168 hourly being 34.75%, 17.03%, 14.06% and 4.46% respectively. The refrigerated semen sample with RPMI however kept for 9 days with motility after 24, 48, 72, 168 and 216 hours being 53.47%, 37.62%, 25.64%, 8.32%

Fig. 14. Effect of refrigeration on motility of semen sample extended with NaCl (left) and RPMI (right). The refrigerated semen sample retained motility till the 7th (in NaCl) and 9th day (in RPMI) whereas semen samples at room temperature only lasted till the first day

production of waste since the sperm cells metabolised at the normal rate.

and 5.25% respectively.

after storage.

The art of refrigeration provides a low temperature which lowers the metabolic rate of living organisms. The extended semen samples maintained at room temperature proceeded at the normal metabolic rate, hence the sperm cells could not survive up to 48 h. When the sperm cells were still within the fish in the testis, they were supplied with nutrients and the waste they produced are excreted out of the testis, they cannot be supplied with energy or nutrients except provided externally as in tissue culture. Their wastes also accumulate in the solution in which they are suspended in.

High metabolic rate means faster rate of using up available resources by living organism such as nutrients and energy. It also means that waste will be produced at a faster rate thereby causing fast accumulation of waste in the solution in which the sperm cells are suspended. This accumulation will immediately reach a toxic level causing fatality in the sperm cells. Whereas the low temperature provided by the refrigerator to the refrigerated semen sample reduced the metabolic rate of the sperm cells, thereby reducing the rate at which the available nutrients and energy in the semen-extender solution are used up. The nutrient and energy in the semen-extender solution lasted a much longer period when refrigerated, thus keeping the sperm cells alive for a longer period than in the semen samples extended at room temperature.
