**5. Acknowledgements**

This research was made possible by research grants from the OAU University Research Committee (11812 AVT), National Centre for Genetic Resources and Biotechnology, Ibadan and the World Bank-Assisted STEP-B (Science and Technology Post-Basic), Abuja. The authors are grateful for the technical assistance of Messrs. Akin Babatunde, Samuel Oladejo, Rotimi Solanke and Wasiu Olaniyi. Our sincere gratitude goes to Prof. B. Solomon of NABDA (National Biotechnology Development Agency) for partially funding the publication charges and Dr. Igor Katkov and Mrs O. Osoniyi for their comments and their expert editing of the manuscript.

#### **6. References**

328 Current Frontiers in Cryopreservation

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

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

clutch of eggs since there is a direct relationship between motility and fertility.

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

be able to induce the spawning of the female broodstock.

**5. Acknowledgements** 

expert editing of the manuscript.

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

This research was made possible by research grants from the OAU University Research Committee (11812 AVT), National Centre for Genetic Resources and Biotechnology, Ibadan and the World Bank-Assisted STEP-B (Science and Technology Post-Basic), Abuja. The authors are grateful for the technical assistance of Messrs. Akin Babatunde, Samuel Oladejo, Rotimi Solanke and Wasiu Olaniyi. Our sincere gratitude goes to Prof. B. Solomon of NABDA (National Biotechnology Development Agency) for partially funding the publication charges and Dr. Igor Katkov and Mrs O. Osoniyi for their comments and their


http://www.Cryobiosystem.imv. com/CBS/Cryobiology/cons.cbs-asp.


**Part 5** 

**Cryopreservation of Plants** 


**Part 5** 

**Cryopreservation of Plants** 

330 Current Frontiers in Cryopreservation

Omitogun, O.G, Olaniyan, O. F., Oyeleye, O.O., Ojiokpota, C. Aladele, S.E. and Odofin, W.

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Riley, K. L. P. (2002). Refrigerated storage and cryopreservation of sperm for the production

SIGMA (1994). Cell counting and cell viability. Saint Louis, MO 63178 USA. Pp. 1634-1635. Steyn, G.J, Van Vuren, J. H., Schoonbee, H.J. and Chao, N. (1985). Preliminary investigations

Steyn, G.J. (1987). The fertilizing capacity of cyopreserved sharptooth catfish, *Clarias* 

Suquet, M., Dreanno C., Fauvel, C., Cosson, J. and Billard, R. (2000) Cryopreservation of

Sydenham, D.H.J. (1980). New species of *Clarias* from West Africa. Rev. Zool. *Afr., Vol* 

Teugels, G.G. (1986a). Clariidae. In: Daget, J., Gosse, J-P., Thys van den Audenaerde,D.F.E.

Teugels, G.G. (1986b). A systematic revision of the African species of the genus Clarias (Pisces:Clariidae). *Annals Musee. Revue. Africain. Contributions*. Vol. 247 pp. 1-199. Tiersch, T. R., Goudie, C.A. and Carmichael, G.J. (1994). Cryopreservation of channel catfish

Urbanyi B., Horvath, A., Varga, Z. and Horvath, L. (1999). Effect of extenders on sperm

Van der Walt, L.D., Van der Bank, F.H. and Steyn, G.J. (1993). The suitability of using

Viveiros, A.T., So, N. and Komen, J. (2000). Sperm cryopreservation of African catfish,

(editors). Check-list of the Freshwater fishes of Africa, Brussels, MRAC, Tervuren,

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*Clarias gariepinu*s; Cryoprotectants, freezing rates and sperm: egg dilution ratio.

sperm in marine fish. *Aquaculture Research* Vol. 31 pp. 231-243.

*Agriculture* Vol. 22 .No.(1) pp.11-16. Ile-Ife, Nigeria. ISSN 0331-6351. Padhi, B.K. and Mandal, R.K. (1995). Cryopreservation of spermatozoa of two Asian

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T.(2010). Potentials of short term and long term cryopreserved sperm of African giant catfish *(Clarias gariepinus* Burchell 1822) for aquaculture. *African Journal of Biotechnology* Vol. 9 No. 41. pp. 6973-6982, ISSN 1684–5315. Academic Journals. Oteme, J., Nunez-Rodriguez, J., Kouassi, C.K. Hom, S. and Agnese, J.F. (1996). Testicular

structure, spermatogenesis and sperm cryopreservation in the African clariid catfish, *Heterobranchus longifilis* (Valenciennes, 1840). *Aquaculture Research* Vol. 27

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**17**

**Comparison of Cryopreservation Methods of** 

There is a trend to preserve the plant germplasm by not only conventional *ex situ* methods or *in vitro* techniques, but also, more recently, by cryopreservation. Cryopreservation techniques are based on the storage of plant samples at very low temperature at which practically no chemical reactions occur and consequently, neither aging nor genetic changes of plant material. There has been a great development progress of cryopreservation methods during last years. Cryopreservation becomes a highly utilized technique for germplasm conservation. Generally the cryopreservation is storage of the samples. The samples can be e.g. organs and shoots tips from *in vitro* culture, or from the field, such as mature, immature bulbils, cloves of garlic or dormant buds of fruit trees, at the ultra-low temperature (mainly

Although the technique was introduced for plants in the '70s, it has never been applied on a wide scale due to the high cost of cryo-freezers; indeed, it was used in order to escape the formation of lethal intracellular ice crystals, time-consuming and laborious slow-cooling procedures. A new cryogenic - vitrification technique is now available, aiming at the direct immersion of plant specimens from tissue cultures in liquid nitrogen, without resorting to an expensive apparatus for slow cooling and with a considerable simplification of the procedures (Benson, 2008). The vitrification method simplifies cryogenic process and makes possible an increased application of cryopreservation on wide-range plant genetic resources. The glassy state is the objective status of cryopreservation methods named vitrification.

The aim of this study is a comparison of different cryopreservation methods based on the vitrification achieved by dehydration and glass transition temperature (Tg), and their efficiency towards optimal regeneration of vegetatively propagated plants. The thermal characteristics, evaluation of frozen water content, and the glass transition temperature were

Some of vegetatively propagated plants are not able to reproduce by seeds e.g. garlic plant (*Allium sativum* L.). The only way how to propagate it is to use its cloves or bulbils

**2. Importance of cryopreservation of vegetatively propagated plants** 

**1. Introduction** 

–196 °C, the temperature of liquid nitrogen).

measured by a differential scanning calorimeter.

**Vegetatively Propagated Crops Based** 

**on Thermal Analysis** 

and Renata Kotková *Crop Research Institute The Czech Republic* 

Jiří Zámečník, Miloš Faltus, Alois Bilavčík
