This document summarizes advances and applications of cryopreservation techniques in fisheries. It discusses the principles and mechanisms of cryopreservation including the use of cryoprotectants and liquid nitrogen storage. Studies on cryopreserving sperm from various fish species like Indian major carps, brown trout, and koi carp are described. Cryopreservation of fish sperm has applications for conservation of genetic resources, selective breeding programs, and sustainable aquaculture.

1. Advances and Applications of
Cryopreservation Techniques in
Fisheries
By- Deepa Bhatt
deepabhatt94@gmail.com
GADVASU

2. CONTENTS
• Introduction
• Need of Cryopreservation
• Principle
• Mechanism of cryopreservation
• Advances & Applications

3. INTRODUCTION
• Cryopreservation is a non-lethal storage
of biological material at ultra-low
temperature
• At the temperature of liquid nitrogen
(-1960C) almost all metabolic activities
of cells are ceased and the sample can
then be preserved in such state for
extended periods

4. Why Cryopreservation?
Cryopreservation has several practical applications in
fisheries. They are :
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 wherein a
large number of families have to be maintained
5. Production of androgenetic fish
6. Conservation of genetic resources

5. 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°)

6. Why Liquid nitrogen ?
• Chemically inert
• Relatively low cost
• Non toxic
• Non flammable
• Readily available
Liquid nitrogen is most widely used
material for cryopreservation.

7. Short term preservation
• Short term preservation of gametes can be done
for some hours to few days at temperature b/w
0-4° C
• Both undiluted and diluted semen of common
carp and rainbow trout could be preserved for
few days at this temperature
• In salmonids, spermatozoa in undiluted semen
survives upto one month at temperatures of
around 1- 4°C with the addition of antibiotics
and oxygen

8. Long term Cryopreservation

9. 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 3-4 hrs

11. 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”
• Extender keeps the spermatozoa alive in an
inactive condition

12. 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. Polyethlene
Glycol or
sachharides)
FUNCTIONS OF
CRYOPROTECTANTS
 Protect cells from ice
crystal damage
 Penetrates into cells
and should have low
toxicity.
 Reduce amount of ice
formed at given
temperature as they
lower freezing point

13. 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

14. 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

15. Freezing phase
The Equilibrated milt-diluent mixture is frozen
at very low temperature
• There are three ways of freezing

16. 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

17. Freezing
It is noted that the
temperature range
between 0 to -40°C is
the most critical
during freezing since
most of the
cryoinjuries takes
place in this
temperature range
When temperature goes
below about -120°C the
freeze concentrated
residual liquid in the
extra- cellular solution
and the dehydrated
cytoplasm virtify and
further cellular damage
does not take place

18. 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

19. It is the process that transforms Intracellular waters to non crystalline
solids after freezing. This occurs under two circumstances
The temperature at which vitrification begins is called as glass
transformation temperature , which is -13°C for water
Vertified cell/ glassy
state
Vitrification
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

20. V

22. 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

23. 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
• This transfer is necessary since the cells get
damaged if left for long in warm (37°C - 45°C) water
The Cryomilt samples of carps can be thawed in
warm water of 38 ± 1ºC for 7-9 seconds

24. 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

25. Viability of Cryopreserved spermatozoa
•Spermatozoa stored under LN2 remain
fertile indefinite
•They should be thawed only when required
for checking motility
•Motility, fertilization, hatching rates and
fry survival, etc. are the common criteria
for judging the post-thaw viability/fertility
of Cryopreserved Spermatozoa

26. Cryopreservation of eggs/embryos
• Not good results. The problems are –
• However, success has been achieved with
invertebrate eggs and embryos
• Sea urchin embryos, oyster larvae (trochophore) and
penaied shrimp naupli have been successfully
cryopreserved
Insufficient dehydration during freezing due to
relatively large size (1-6 mm) of fish eggs
The presence of membranes of different water
permeability

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

28. 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
• Successful cryopreservation of fish sperm have
been achieved for >200 fish species
• Success of cryopreservation mainly depends on
the prevention of cryoinjury during freezing and
thawing
First successful cryopreservation of fish sperm was
reported in 1950s

29. Fish sperm cryopreservation assists conservation of fish
biodiversity through gene banking of endangered
species, and assists aquaculture by providing flexibility
in spawning of females and selective breeding
through
• The technique also ensures preservation of genetic
materials of superior wild fish populations and
enables gene transfer from wild and hatchery stocks
(Cloud et al. 1990, Tiersch et al. 1998)
Applications in Aquaculture
Synchronizing artificial reproduction,
Efficient utilization of semen
Maintaining genetic variability of broodstocks (Tiersch 2000,
Lahnsteiner 2004).

30. Endangered

31. • 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%)
Studies were conducted on cryopreservation of spermatozoa
from Indian major carps.

32. • It can be concluded that the cryopreservation protocol
developed is rather effective and 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)

33. Cont..
• In addition, it is possible to suggest that sperm
cryopreserved with ionic extender containing
10% DMSO packed in 0.25 mL volume straws
and thawed at 30°C is the most suitable
conditions
• As it retain the sperm quality in koi carp having
Optimal sperm motility
Duration of motility as well as
High fertility percentages close to the values
obtained with fresh sperm. (Yusuf Bozkurt, İlker
Yavas, and Fikret Karaca)

34. Fish species Method Diluent
Cryoprotectant Extender
References
Yellowfin bream vial-LN 10% glycerol 300 mo SW Thorogood &
Blackshaw 1992
Grouper vial-LN vapor 10% DMSO OH-251 Withler & Lim
1982
Grey Mullet straw-LN vapor 5-10% glycerol Ringer solution
for marine fish
Chao et al. 1975
Bluefin Tuna straw-LN vapor 12.5% DMSO
12.5% glycerol
10% glucose Doi et al. 1982
Cobia vial-methanol-dry 10% DMSO+10%
yolk
3 mM glucose Caylor et al. 1994
Summary of protocols used for cryopreservation of marine fish sperm

35. s
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

36. IPS - Induced Pluripotent Stem
Advances of Cryopreservations

38. • Dramatic decline in fish populations necessitates
urgent action to enable gametes and embryo
cryopreservation as an aid to conservation
• High yolk content and low membrane
permeabilities have frustrated their successful
cryopreservation, by limiting water removal and
cryoprotectant penetration

39. (i) Permeabilisation of embryo membranes, through
media modification and ultra-sound treatment
(ii) Direct modification of the yolk mass by micro-
manipulation, and
(iii) The use of impedance spectroscopy for rapid
assessment of embryo membrane permeability
• The approach to fish Oocyte Cryopreservation has
been directed at optimizing low toxicity Cryoprotectant
mixtures, and their use in protecting oocytes at a range
of sub-zero temperatures
Research on fish embryo cryopreservation is currently
focused on the three new approaches:

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. Opens the door for rapid Genetic improvement.
• 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.
The Future Prospects for Application of
Cryopreservation in Aquatic species