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 1st 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. a) 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).
b) Chromosome set manipulation - Manipulation of chromosome-sets
(polyploidization) has been accomplished for many aquatic species
through thermal and chemical shocks to developing embryos.
c) 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. d) 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.
e) 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.
f) 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. 1. Wider distribution of gametes from one
location to another location
2. Reduces number of male broodfish to be
maintained
3. Facilitates extension of period of seed
Availability
4. Selective breeding programmes where in a
large number of families have to be maintained
5. Production of androgenetic fish
6. Conservation of genetic resources
Cryopreservation has several practical applications in
fisheries. They are :
WhyCryopreservation?

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
Collected milt samples are kept in 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,Cacl2 etc.

15. CRYOPROTECTANTS
Penetrating
(Intracellular)
Non Penetrating
(Extracellular)
Penetrating 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
and should have
cells
low
toxicity.
 Reduce amount of ice
formed at
temperature as
given
they
lower freezingpoint

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
McAndrew,
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. During freezing several physico- chemical changes takes
place within the cell and its surrounding area
Initially ice crystal formation occurs in the extra cellular
medium due to freezing.
As a result, the extra-cellular medium becomes
hypertonic to the cell
To maintain the osmotic balance the intra- cellular water
comes out leading to the reduction of the cellular volume
These changes cause mechanical damage to the cell
Freezing

19. There are two potential sources of cell damage
during Cryopreservation.
1. Formation of large ice
crystals inside the cell
2. 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
1. If the cooling rate is very high, it does not allow
sufficient time for the water molecules to crystallize
2. Solution is so concentrated that the high viscosity at
low temperature does not allow water molecules to
crystallized
• The temperature at which vitrification begins is
called as glass transformation temperature ,
which is -13°C for water

21. Storage
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
1953 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. Studies were conducted on cryopreservation of spermatozoa
from Indian major carps.
Effect of different concentration of glycerol 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
Brown
trout
6 Erdahl
and
Graham
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
Striped
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 2Me2So & 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.  Improvement of existing hatchery
operations by providing sperm on demand
 Efficient use of facilities & create new
opportunities in the hatchery.
 Endangered species, research models, 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,

43. 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)

44. 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+car
p+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.

45. 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
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• Franěk, R., Marinović, Z., Lujić, J., Urbányi, B., Fučíková, M., Kašpar, V., Pšenička,
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• Shaliutina-Kolešová, A., Dietrich, M., Xian, M. and Nian, R., 2019. Seminal
plasma transferrin effects on cryopreserved common carp Cyprinus carpio
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• 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
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46. • Hagedorn, M.M., Daly, J.P., Carter, V.L., Cole, K.S., Jaafar, Z., Lager, C.V. and
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cells via germ cell transplantation. Stem cell research, 29, pp.103-110
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Conti,

47. THANK YOU