Cryopreservation and its application to aquaculture.pptx

What is Cryopreservation ?
Cryopreservation is a process where biological materials such as cells and tissues are preserved by cooling to very low temperatures, usually at -196°C (the temperature of liquid nitrogen), yet remain viable after later warming to temperatures above 0°C.

Cryopreservation in aquatic species goes back 65 years and began about the same time as similar research was performed in livestock (Blaxter 2011).

In India, NBFGR & CIFA are the primary organization carrying out fish sperm cryopreservation for long term gene banking (J. K. Jena 2012)

1. Cryopreservation and its Application to Aquaculture Narsingh Kashyap, MFSc 1 st year Department of Fish Genetics & Breeding ,IFPGS, Vaniyanchavadi, Chennai

2. Introduction Cryopreservation Principle of cryopreservation Application in Aquaculture Current Status Future Prospects Conclusion Reference Fig.- Cryocan

3. Genetic improvement   occurs when the  genetic  merit is improved through Several Methods. The  improvement  in  genetic  merit refers to the overall  improvement  in a flock brought about by selection for a number of traits that contribute to the flock's breeding objective. Genetic improvement  is a field that can contribute to sustainable aquaculture by  improving  traits like Growth, Flesh quality, Disease resistance, Feed Conversion ratio, Age of sexual maturation & Fecundity of individuals. Introduction

4. Hybridization & Crossbreeding - Crossbreeding and hybridization can be utilized to combine favourable qualities from two genetically different groups and to take advantage of hybrid vigour (heterosis). Chromosome set manipulation - Manipulation of chromosome-sets ( polyploidization ) has been accomplished for many aquatic species through thermal and chemical shocks to developing embryos. Sex manipulation - Manipulation of sex can be of advantage in species with sexual dimorphism in important traits. It Also help to produce monosex population. Method of Genetic improvement

5. Genetic engineering - is a broad heading that includes chromosome set manipulations and transgenesis . Chromosome set manipulation is a group of techniques in which sets of chromosomes in organisms are modified. Selective breeding - The basic tool available for genetic improvement, selective breeding, entails choosing the animals with the highest genetic value as breeders for the next generation. Cryopreservation - Cryopreservation is the technique by which living cells/tissues / gametes are preserved at very low temperature usually at -196 °C. CONT..

6. What is Cryopreservation ? Cryopreservation is a process where biological materials such as cells and tissues are preserved by cooling to very low temperatures, usually at -196°C (the temperature of liquid nitrogen), yet remain viable after later warming to temperatures above 0°C. Cryopreservation in aquatic species goes back 65 years and began about the same time as similar research was performed in livestock ( Blaxter 2011). In India, NBFGR & CIFA are the primary organization carrying out fish sperm cryopreservation for long term gene banking (J. K. Jena 2012)

7. Wi d e r di s tribution o f g a m e t e s f r om one location to another location R educ e s number o f mal e b r o o d f ish t o be maintained 3 . F acil i ta t e s e x t e nsion o f peri o d o f seed Availability 4 . Selec t i v e b r eed i ng p r o g r a m m e s whe r e in a large number of families have to be maintained Production of androgenetic fish Conservation of genetic resources Cr y o p r es e r v a tion h a s s e v e r al p r a c ti c al a p p l i c a tions in fisheries. They are : Why Cryopreservation?

8. Principle of Cryopreservation Principle - To bring cells or tissue to a zero metabolism and non dividing state by reducing the temperature in the presence of cryoprotectant (anti freeze). It can be done over : Solid carbon dioxide (-79 ° ) Low temperature deep freezer (-80 ° ) Vapour phase nitrogen (-150 ° ) Liquid nitrogen (-196 ° )

9. Liquid nitrogen is most widely used material for Cryopreservation Why Liquid Nitrogen ? Chemically inert Relatively low cost Non toxic Non flammable Readily available

10. Non-Cryogenic preservation ( Short term) Short term Preservation of gametes can be done for 3-4 days at Temperature between 0 - 4 °C. The milt sample is diluted with extender and stored in a thermocol chamber with ice or in refrigerator. Both undiluted and diluted semen of common carp and rainbow trout could be preserved for few days at this temperature .

11. Long term Cryopreservation

12. Pre freezing phase Collection of spermatozoa from mature male, avoiding contamination with urine, mucus, water, faeces, etc Males may be injected with spawning agent to ensure higher milt volume Milt is collected by hand stripping into an ice cold sterilized tube Collec t e d mil t s a mple s a r e k e pt i n a refrigerator After a gap of 4 - 6 hrs

13. Conti,,

14. EXTENDER For preservation, milt samples are diluted in a slightly hypertonic electrolyte medium termed as extender. Extender is as “a solution of salts, sometimes including organic compounds, which helps maintain viability of cells during refrigeration”. It also inhibits the activation of spermatozoa and functions as a medium for cryoprotectant. Ex.- Nacl, Kcl,Cacl 2 etc.

15. CRYOPROTECTANTS Penetrating (I n t r a c ellu l ar) Non Penetrating (Extracellular) P en e t r a t i ng the cell membrane and enter into Cytocol (E.g. DMSO, Glycerol, Methanol, Ethylene, Glycol ) Do not Penetrate the cell membrane (E.g. dextran ,Glucose, Sucrose or sachharides ) FUNCTIONS OF CRYOPROTECTANTS Protect cells from ice crystal damage Penetrates into a n d s ho u l d h av e c e l ls l o w toxicity. Reduce amount of ice f ormed at t e mp e r at u r e as gi v e n t h e y lower freezing point

16. Species Extender Cryoprotectant References European catfish 200mM NaCl 12% Glycerol Linhart et al , 1993 Lates calcarifer Ringers solution & 20% egg yolk 5% DMSO or 20 Glycerol Leung, 1987 Tilapia Modified Ringers solution 12.5% Methanol Rana and McAndre w , 1989 Channel catfish hank’s balance salt solution 5 or 10 % Methanol Tiersch et al ., 1994 Salmonid fishes 7 % egg yolk, 0.5% sucrose % DMSO + 1% Glycerol Lahnsteiner et al ., 1995 Striped bass 0.6% NaCl 10% Glycerol Padhi and Mandal, 1995 Extender & Cryoprotectant combination used for spermatozoa preservation in some fishes (P. C. Thomas, Breeding & Seed production of finfish & shellfish)

17. Equilibration of milt diluent mixture The milt dilution mixture is kept at low temperature for equilibration In case of Indian major carps equilibration period can be 45 minutes The low temperature reduces the toxicity of the Cryoprotectant on cell, as Permiability is reduced at low temperature for most chemical like DMSO

18. Durin g f r e e zi n g se v e r al p h ys i c o - ch e mica l ch a n g e s t a k es place within the cell and its surrounding area Initia l ly i c e c r y s t al f orm a tion oc c u r s i n the e x t r a cellular medium due to freezing. A s a r esult, t h e e x t r a - c el l ular m e di u m be c omes hypertonic to the cell T o mai n t a in the os m otic balan c e the i n t r a - c el l u lar w a t er comes out leading to the reduction of the cellular volume These changes cause mechanical damage to the cell F r e e zing

19. There are two potential sources of cell damage during Cryopreservation. Formation of large ice crystals inside the cell Intracellular concentration of solutes increase to toxic levels before or during freezing as a result of dehydration

20. Vitrification It is the process that transforms Intracellular waters to non crystalline solids after freezing. This occurs under two circumstances I f t h e c o o l ing r at e i s v e r y high, i t do e s not al l ow sufficient time for the water molecules to crystallize S o l u t i on i s so c o n c e n t r a t e d th a t th e h i g h viscosit y a t low temperature does not allow water molecules to crystallized Th e t emp e r a tu r e a t whi c h vitri f i c a tion be g ins is c alled as glass transformation temperature , which is -13°C for water

21. S t o r a g e To manage Cryobanks efficiently, it is essential to keep comprehensive records of all stocks preserved Storage is ideally done in liquid nitrogen refrigerator at -150°C in vapor phase or at -196°C in the liquid phase

22. Thawing Thawing is done by putting the vial/ampoule containing the sample in a warm water bath (35°to 45°C ) . As the thawing occurs, (ice completely melts the ampoule) are quickly transferred to water bath at temperature 20 to 25°C . The Cryomilt samples of carps can be thawed in warm water of 38 ± 1ºC for 7- 9 seconds

23. Insemination and post insemination The thawed milt is mixed properly to the stripped eggs immediately and activated by a drop of water Percentage of fertilization should be determined to evaluate gamete quality The fertilized eggs are to be incubated in flow through system so as to remove the cryoprotectant trace from the eggs as it is toxic to the developing embryo

24. Cryopreservation of eggs/embryos Not good results. The problems are – Insufficient dehydration during freezing due to relatively large size (1-6 mm) of fish eggs The presence of membranes of different water permeability However, success has been achieved with invertebrate eggs and embryos .

25. Cryopreservation of eggs/embryos To Successfully Cryopreserve an embryo, an osmotically active water must exit the cells and an appropriate cryprotectant must enter the cells

26. Applications in Aquaculture To preserve and store both maternal and paternal gametes provides a reliable source of fish genetic material for scientific and aquaculture purposes as well as for conservation of biodiversity Its Open the door for rapid genetic improvement. Fish sperm cryopreservation assists conservation of fish biodiversity through gene banking of endangered species. First successful cryopreservation of fish sperm was reported in 195 3 by Blaxter

27. Broodstock maintenance is simplified: off-season spawning can be induced only in females and cryopreserved sperm can be used to fertilize the eggs. Through the Cryopreservation , Genetic materials (Gametes) can carry throughout the world and initiate different breeding programme It reduce the cost of Broodstock management. Efficient utilization of semen

28. St u d i e s w e r e c on d uc t e d on c r y o p r es e r v a t i on of sperm a t o z oa from Indian major carps. Effect o f d i f f e r e n t c onc e n t r a t i on o f gl y c e r ol and Equilibration periods on the post thaw motility of spermatozoa from Catla, Rohu and Mrigal were observed The maximum motility (80 – 85%) was observed with the equilibration period of 20 – 40 minutes with the concentration of 10-15% of glycerol . Rohu showed the same trend with the maximum motility of 78- 87%. In Mrigal maximum motility (85-88%) was observed with the equilibration time of (20-40 minutes) with the concentration of glycerol in between (10-15%)

29. It can be concluded that the cryopreservation protocol developed is rather effective of brown trout ( Salmo trutta ) and Ornamental koi carp ( Cyprinus carpio ) sperm can be successfully cryopreserved It seems that cryopreservation of brown trout sperm with ionic extenders containing 15% egg yolk is rather effective on post-thaw sperm quality (Yusuf Bozkurt, İlker Yavas, and Fikret Karaca)

30. This study Investigate sperm quality of fresh (control) and thawed of great scallop. Sperm quality was assed using 4 parameter of fresh & thawed sperm sample. Parameter Techniques used Sperm motility Computer assisted sperm quality plugin Intracellular ATP content ATP like Kit Sperm integrity Flow cytometery Sperm morphology Transmission electron microscope

31. Conti.. Significance decrease of both the percentage of motile spermatozoa ( reduction 75%) in thawed spermatozoa & (86%) observed in Fresh spermatozoa. The living spermatozoa in thawed spermatozoa is (72.4 +- 2.5) & compare to (86.4 +- 1.1) in fresh sample. There is no significance different of intracellular sperm ATP count & no morphological difference. In conclusion the parameter studies show the quality of thawed great scallop sperm was lower than that of fresh cells but was still sufficient for use in aquaculture programme .

32. Species Extender Extender composition Dilution ratio Zebrafish BSMIS 75 mM NaCl, 70 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM Tris Testicular sperm 1 : 9 B r o w n trout 6 Erdahl and G r aham 99.95 mM NaCl, 0.52 mM citric acid, 55.51 mM glucose, 2.26 mM KOH, 10% egg yolk 1 : 5 Common carp --- 3500 mM glucose, 30 mM Tris 1 : 9 Silver carp --- 68.38 mM NaCl, 27.2 mM sodium citrate, 11.01 mM glucose 1 : 2 African catfish Ginsburg fish Ringer 123.2 mM NaCl, 3.75 mM KCl, 3 mM CaCl2, 2.65 mM NaHCO3 Testicular sperm 1 : 9 Strip e d bass --- 239.56 mM NaCl, 5.36 mM KCl, 23.81 mM NaHCO3, 5.55 mM glucose, 75 mM glycine 1 : 3 Successful Cryopreservation of fish sperm have been achieved for more than 200 fish species.

33. In Practice , no significant change of biological importance occur below -150 °C & there for materials can be stored in liquid nitrogen vopar or liquid nitrogen -196 °C.

34. The Testing resulting in the higher survival achieved using 2Me 2 So & Cooling rate of -1°C / min. The functional of cryopreserve cells was tested by interspecific transplantation into sterilized gold fish recipients.

35. Semen was Diluted with Kurokuro extender ( composing 180 mM Nacl , 1.36 mM Cacl2,2.38mM, NaHco3 and 10% DMSO) Cryopreservation medium was supplement with plasma transferrin, bovine serum albumin or anti freeze protein type I & III. It conclude that incorporation of Transferrin in the extender before freeze improve cryopreservation of common carp spermatozoa where as supplementation with AFP III in greater concentration was more effective,

36. The alteration of sperm molecules ( Including DNA, Phospholipids, & Protein ) During the cryopreservation is at least partially responsible for reduced Spermatozoa function and quality. The molecular basis of protective mechanism of these protein during cryopreservation still unclear.

37. Current Status of Cryopreservation in Aquaculture To date milt of over 200 Sps . Of Fresh water and Marine fish have been Cryopreserve ( Lakra et al. 1993) Cryopreservation has not yet fully adopted for fish because fish egg are relatively large & rich in lipid & egg yolk ( Yoshizaki et al. 2019) Cryopreservation of fish tissue especially in fin pieces can easily be considered for cryobanking with some minor technical (S M Paramo et al. 2017)

38. IPS - Induced Pluripotent Stem Advances of Cryopreservations

39. The storage of common carp Embryo was possible for up to 7 days. Combination of methanol with propylene glycol gave higher survival rate after 1 day & 7 days stored at -2°C.

40. Imp r o v e me n t o f e xi s ting h a t ch e ry operations by providing sperm on demand E f ficie n t us e o f f acili t i es & c r e a t e n e w opportunities in the hatchery. Endan g e r e d sp e cies, r es e a r ch mode l s , or improved farmed strains can be protected. The Future Prospects for Application of Cryopreservation in Aquatic species

41. Frozen sperm can be used in breeding programs to create new improved lines. Cryopreserved sperm of aquatic species, within the coming decade, become an entirely new industry itself. The global market for livestock sperm is around a billion dollars each year. Conti,

42. Conclusion We can established seed bank , cryo bank for Endangered or Threatened ,wild strains or commercial importance species (Continued effort) Cryopreservation of fish eggs or embryos is still not possible due to there large size & high yolk content (need of focussed research) Undifferentiated germ cells, such as primordial germ cells, Spermatogonia & oogonia as materials for cryopreservation (Prioritization)

43. Reference P C THOMAS et al., Breeding and Seed Production of FIN FISH & SHELL FISH A E Eknath, Utilization of Cryomilt in Aquaculture ,ICAR-CIFA Trygve Gjedrem , Selection and Breeding Programs in Aquaculture AKVAFORSK , Institute of Aquaculture Research AS, Norway. https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=Cryopreservation+of+carp+spermatozoa&oq= https://en.wikipedia.org/wiki/Cryopreservation Basic Principles of Cryopreservation fao http://www.fao.org/3/i3017e/i3017e00.htm https://en.wikipedia .org/wiki/ Vitrification Liu, Y., Blackburn, H., Taylor, S.S. and Tiersch , T.R., 2019. Development of germplasm repositories to assist conservation of endangered fishes: Examples from small-bodied livebearing fishes.  Theriogenology ,  135 , pp.138-151. Herrero , L., Martínez , M. and Garcia-Velasco, J.A., 2011. Current status of human oocyte and embryo cryopreservation.  Current Opinion in Obstetrics and Gynecology ,  23 (4), pp.245-250. Kopeika , E., Kopeika , J. and Zhang, T., 2007. Cryopreservation of fish sperm. In  Cryopreservation and freeze-drying protocols  (pp. 203-217). Humana Press.

44. Conti, Francisco Marco jimenez & Hiilya Akdemir ,cryopreservation in Eukaryotes Patra , S., et al, (2016). Transplantation worthiness of cryopreserved germ cells of Indian major carp rohu , Labeo rohita.  Jena, J.K. and Gopalakrishnan , A., 2012. Aquatic biodiversity management in India.  Franěk , R., Marinović , Z., Lujić , J., Urbányi , B., Fučíková , M., Kašpar , V., Pšenička , M. and Horváth , Á., 2019. Cryopreservation and transplantation of common carp spermatogonia .  PloS one ,  14 (4). Shaliutina-Kolešová , A., Dietrich, M., Xian, M. and Nian , R., 2019. Seminal plasma transferrin effects on cryopreserved common carp Cyprinus carpio sperm and comparison with bovine serum albumin and antifreeze proteins.  Animal reproduction science ,  204 , pp.125-130. Xin , M., Niksirat , H., Shaliutina‐Kolešová , A., Siddique , M.A.M., Sterba , J., Boryshpolets , S. and Linhart , O., 2019. Molecular and subcellular cryoinjury of fish spermatozoa and approaches to improve cryopreservation.  Reviews in Aquaculture .

45. Hagedorn , M.M., Daly, J.P., Carter, V.L., Cole, K.S., Jaafar , Z., Lager, C.V. and Parenti , L.R., 2018. Cryopreservation of fish spermatogonial cells: the future of natural history collections.  Scientific reports ,  8 (1), pp.1-11. Yoshizaki , G. and Lee, S., 2018. Production of live fish derived from frozen germ cells via germ cell transplantation.  Stem cell research ,  29 , pp.103-110 Adams, S.L., Smith, J.F., Roberts, R.D., Janke , A.R., King, N.G., Tervit , H.R. and Webb, S.C., 2008. Application of sperm cryopreservation in selective breeding of the Pacific oyster, Crassostrea gigas (Thunberg).  Aquaculture Research ,  39 (13), pp.1434-1442 Keivanloo , S., Sudagar , M. and Mazandarani , M., 2019. Evaluating the suitability of cryopreservation solutions for common carp ( Cyprinus carpio ) embryos stored at-2° C.  Iranian Journal of Fisheries Sciences ,  18 (4), pp.1035-1045. Liu, Y., Blackburn, H., Taylor, S.S. and Tiersch , T.R., 2019. Development of germplasm repositories to assist conservation of endangered fishes: Examples from small-bodied livebearing fishes.  Theriogenology ,  135 , pp.138-151. Conti,

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