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

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

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

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 gariepinus*.

Cryopreservation of the Sperm

different extenders.

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 325

Fig. 16. Effect of the different extenders on the motility of the sperm cells of *Clarias gariepinus.*

Fig. 17. The decline in motility as the period of storage of refrigerated sperm increased in

Fig. 15. Effect of osmolality on the sperm motility of refrigerated semen sample in Ca-F HBSS The 200mOsmol/kg Ca-F HBSS extended semen sample retained motility up to day 12 (0.5%)

#### **3.5.3 Effect of extenders on % motility**

The refrigerated semen sample with the RPMI culture solution had relatively very high motility at 24 hours after refrigeration; it had a motility of 53.47% as against the 34.85% motility of the semen sample with the 200mOsmol/kg Ca-F HBSS which comes next in rank with it after 24 h. The RPMI extended semen sample also had the highest motility after 48 h of refrigeration (37.62%) with the next in rank being semen sample extended with 300mOsmol/kg Ca-F HBSS with motility of 30.10%. However, the RPMI extended semen sample retained motility only till the fifth day with motility at the fifth day being 5.25% (Fig.16).

The control experiment being semen samples extended with 0.9% NaCl solution retained motility for 7 days with motility at the seventh day being 4.46% and its motility at 24 hours was 34.75% which is exceeded by the semen sample extended with the 200mOsmol/kg Ca-F HBSS which had 34.85% motility at 24 h (Fig. 17).

The relatively high motility retained by the refrigerated semen sample with RPMI culture medium may be due to the additional nutrient supply provided by the RPMI culture medium solution. The RPMI medium culture contains many amino-acids, vitamins and growth factors.

Fig. 15. Effect of osmolality on the sperm motility of refrigerated semen sample in Ca-F HBSS The 200mOsmol/kg Ca-F HBSS extended semen sample retained motility up to day

The refrigerated semen sample with the RPMI culture solution had relatively very high motility at 24 hours after refrigeration; it had a motility of 53.47% as against the 34.85% motility of the semen sample with the 200mOsmol/kg Ca-F HBSS which comes next in rank with it after 24 h. The RPMI extended semen sample also had the highest motility after 48 h of refrigeration (37.62%) with the next in rank being semen sample extended with 300mOsmol/kg Ca-F HBSS with motility of 30.10%. However, the RPMI extended semen sample retained motility only till the fifth day with motility at the fifth day being 5.25%

The control experiment being semen samples extended with 0.9% NaCl solution retained motility for 7 days with motility at the seventh day being 4.46% and its motility at 24 hours was 34.75% which is exceeded by the semen sample extended with the 200mOsmol/kg Ca-F

The relatively high motility retained by the refrigerated semen sample with RPMI culture medium may be due to the additional nutrient supply provided by the RPMI culture medium solution. The RPMI medium culture contains many amino-acids, vitamins and

12 (0.5%)

(Fig.16).

growth factors.

**3.5.3 Effect of extenders on % motility** 

HBSS which had 34.85% motility at 24 h (Fig. 17).

Fig. 16. Effect of the different extenders on the motility of the sperm cells of *Clarias gariepinus.*

Fig. 17. The decline in motility as the period of storage of refrigerated sperm increased in different extenders.

Cryopreservation of the Sperm

profit for the institute (cryobank).

inclusion and higher cost of DMSO.

DGP and DP proved to be the best even for both trials.

are also assured of the viability of the sperm cells they are buying.

for hatchability and fertility in the first trial but only fertility in the second trial.

**4. Conclusion** 

of the African Catfish for the Thriving Aquaculture Industry in Nigeria 327

DGP, GP and GF. Though, they were not all procured from the same source, all gave almost similar total cost which ranges between N7,192. 00 – N7,280. 00 and cost/vial ranges between N 89.00 and N 91.00 (1 \$ = N 150.00; N, naira is the Nigerian currency). Consequently, the proposed economical selling price (in case a cryobank in the University or the collaborating research agency is to be established) considering the present inflation rate is the same for the

The selling price for GF is expected to be reduced irrespective of its total cost because of lower viability rate of semen cryopreseved with it. Profit per vial was also calculated which is highest for GP at N100/vial and lowest for GF at N95/vial. The selling price ranges from N95.00 – N100.00 which would encourage the buyers to buy it affordably with reasonable

However, the total cost of GP and GF are lower because of the lower cost price of glycerol, but GF gave the least total cost because of a slight difference in the type and amount of chemical used. Total cost of DGP is higher than that of DP, because of cost of glucose

From this study it can be concluded that at further dilution of semen together with different cryoprotecting agents, viability of the semen is still maintained but it varies depending on the type of cryoprotecting agents used. DMSO-dimethylsulphoxide proved to be more efficient than other cryopreservatives in preserving sperm viability. Its potential in cryopreservation can be increased when used in combination with a 5% glucose solution, i.e.

Also, viability of African catfish *C. gariepinus* semen cryopreserved in liquid nitrogen can be maintained for a relatively long time provided ideal protocols are strictly followed. From economic feasibility perspective of cryopreservation of catfish semen, cryopreserved semen is economically feasible and profitable for the cryobank institute or company. The farmers

The ability of the African giant catfish (*Clarias gariepinus* Burchell, 1822) semen cryopreserved from 4-8 months with different combinations of extender and cryoprotecting agents, dimethylsulphoxide (DMSO) and glycerol with two extenders: GFR (Ginzburg fish ringer) and PBS (Phosphate buffer saline) to fertilize various egg clutch weights were investigated to evaluate the optimum clutch of egg a milliliter of cryopreserved semen can fertilize. DMSO + glucose with PBS and DMSO+ PBS only proved to be the best cryoprotectant-extender combination in maintaining viability of catfish semen. The optimum viability of the semen was also observed at 4.0-5.0 g of clutch of eggs/ml of semen with little deviation. The first trial was on dilution ratio of 1:1 but in the second trial, the semen was diluted further at a ratio of 1:20 and tested on various egg clutch weights (1, 2, 3, 4, 5 g) to evaluate the viability of cryopreservation even at further dilution. There was a significant effect of different cryoprotecting agents (p<0.05) on egg clutch weights. There was a significant effect (p<0.05)

In another experiment we tried higher dilution ratio 1:40 and with bigger clutch of eggs (120g) of a standard female breeder (1.0 kg + 0.2 kg) simulating the practices of many commercial farmers, this time with the cryopreserved sperm. Compared with the control

type of cryoprotectant to be used, but net profit per vial is slightly different.

#### **3.6 Cost of production of cryopreserved catfish sperm for the aquaculture industry**

The cost analyses for each reagent and materials used in cryopreservation per ml, g or piece is shown in Table 5. Table 6 shows the cost estimates for the 4-best cryoprotecting agents (DP,


Table 5. The cost of reagents and other consumables used to cryopreserve African catfish semen in liquid Nitrogen (LN2) and listed and the rates per l or ml, g, or per piece are estimated


Estimation based on 200 ml extender for each cryoprotective agent: DP:DMSO-PBSS, DGP: DMSO-Glucose-PBS; GP: Glycerol-PBS; GF: Glycerol-Fish Ringer .

Table 6. Cost estimates/ml in Naira (N) for preparation of cryopreserved African catfish semen using different cryoprotectants

DGP, GP and GF. Though, they were not all procured from the same source, all gave almost similar total cost which ranges between N7,192. 00 – N7,280. 00 and cost/vial ranges between N 89.00 and N 91.00 (1 \$ = N 150.00; N, naira is the Nigerian currency). Consequently, the proposed economical selling price (in case a cryobank in the University or the collaborating research agency is to be established) considering the present inflation rate is the same for the type of cryoprotectant to be used, but net profit per vial is slightly different.

The selling price for GF is expected to be reduced irrespective of its total cost because of lower viability rate of semen cryopreseved with it. Profit per vial was also calculated which is highest for GP at N100/vial and lowest for GF at N95/vial. The selling price ranges from N95.00 – N100.00 which would encourage the buyers to buy it affordably with reasonable profit for the institute (cryobank).

However, the total cost of GP and GF are lower because of the lower cost price of glycerol, but GF gave the least total cost because of a slight difference in the type and amount of chemical used. Total cost of DGP is higher than that of DP, because of cost of glucose inclusion and higher cost of DMSO.
