**4. Conclusion**

326 Current Frontiers in Cryopreservation

**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

**Ingredients DP DGP GP GF**  Broodstock 1200 1200 1200 1200 Liquid Nitrogen 5000 5000 5000 5000 Glycerol **- -** 1.00 1.00 NaCl 5.76 5.76 5.76 5.76 KCl 0.0176 0.0176 0.0176 0.0176 Na2HPO4 1 .564 1 .564 1 .564 **-** KH2PO4 0.208 0.208 0.208 **-** CaCl2 **- - -** 0.2904 NaHCO3 **- - -** 0.5546 Distilled Water 50.00 50.00 50.00 50.00 Glucose **-** 4.10 **- -** Cover slip 1.75 1.75 1.75 1.75 Cryovials (80 paces) 680.00 680.00 680.00 680.00 Saline Solution 3.24 3.24 3.24 3.24 Miscellaneous 300.00 300.00 300.00 300.00 TOTAL 7279.5396 7293.6396 7243.5396 7192.1214 Cost/unit of 80 cryovials 7279.5396 7293.6396 7243.5396 7192.1214 Cost per 1ml cryovial N 90.9942 N 91, 0455 N 90.5442 N 89.9015 Proposed Selling price N 100.00 N 100.00 N 100.00 N 100.00 Profit /cryovial N 9.00 N 8.95 N 9.46 N 11.01 Estimation based on 200 ml extender for each cryoprotective agent: DP:DMSO-PBSS, DGP: DMSO-

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

Glucose-PBS; GP: Glycerol-PBS; GF: Glycerol-Fish Ringer .

semen using different cryoprotectants

**ITEMS COST (in Naira) RATE** Liquid Nitrogen LN2 N16000/20 l Dewar N 800/l DMSO N 3700/100/ml N 37/ml NaCl N 1800/500g N 3.6/g KCl N 2200/500g N 4.4/g Na2HPO4 N 3400/500g N 6.8/g KH2PO4 N 2600/500g N 5.2/g Distilled water N100/l N 0.1/ml NaHCO3 N 6028/500g N 12.056/g CaCl2 N 2200/500g N 4.4/g Glycerol N 2500/2.5l N 1.00/ml Glucose N 4,100/500g N 8.2/g Cover Slip N175/box (100pcs) N 1.75/slip Cryovials N 8500/1000 N8.50/tube Broodstock (male) N 1200 N 1200

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

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

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

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

Cryopreservation of the Sperm

pp. 55-60.

70.

Ibadan. Pp 335-343. ISSN 0189-9686

Vol. 9 Hors Series, pp. 35-41. Coppens International. (2009). www.coppens.eu Cryobiosystems. (2006). Fundamentals of cryobiology-

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breeding of the catfish *Heterobranchus longifilis* Val. (Clariidae) reared in Ebrien

quantity of ova after HCG-induced ovulation in the African catfish, *Heterobranchus longifilis* (Teleostei: Clariidae). *Aquatic Living Resources.* Paris Vol. 8 pp. 309-316. Lubzens E., Daube, N., Pekarsky, I.., Magnus, Y., Cohen, A., Yusefovich, F. and Feigin, P.

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(2006). Potentials of short-term cryopreserved sperm of the giant African catfish*, Clarias gariepinus*( Burchell, 1822) for aquaculture in Nigeria. In: Olakojo, S.A., Ogunbodede, B.A. and Akande, S.R (Eds) Proceedings of the 31st*Annual Conference of the Genetic Society of Nigeria* pp. 141-146. NACGRAB, Moor Plantation, Ibadan,

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fresh semen that gave the highest motility at appreciably high percentage (71%) which was significantly different (P<0.05) from cryopreserved semen diluted at ratios 1:1 (50.52%) and 1:40 (49.05%). 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.

In order to assist subsistence fish farmers who may not be able to obtain cryopreserved semen, the motility of sperm cells stored under refrigerated conditions in different extenders was studied. This research 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 cells 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.* 

The viability of sperm preserved under ordinary refrigerated conditions is possible for a short period of time of 2-7 days depending on the amount of extender used. A culture medium like RPMI 1640 used in this study may give longer life span for sperm cells under refrigerated conditions. Extenders like RPMI 1640 or the cheaper Ca-F HBSS is an alternative that can be recommended for farmers who may have excess of sperm cells from slaughtered male fish for more female gravid eggs that can be sourced within a time period of one week.

The cost of production of a cryotube of sperm (cost of materials, reagents, liquid nitrogen, etc.) was carried out to determine the cost of a milliliter of cryopreserved sperm with a view to selling cryopreserved semen by the research laboratory to farmers who may not be able to afford to buy a male broodstock yielding an affordable cost of N100/ml compared to current cost of a male breeder which is N 1000 -1500 each (1 \$ = 165 N). This also ensures the farmer that the cryopreserved sperm cells they might alternatively buy are viable and will be able to induce the spawning of the female broodstock.
