Cryopreservation of reproductive products of many aquatic species has been successfully achieved. ... Cryopreservation technology applied to the preservation of fish gametes in aquaculture plays an important role in seed production, genetic management of broodstock and conservation of aquatic resources

1. Ashish sahu

2. CONTENT
 INTRODUCTION
 HISTORY OF CRYOPRESERVATION
 OBJECTIVE OF CRYOPRESERVATION
 MILT COMPOSITION AND SPERM QUALITY
OF TELEOSTS
 PRINCIPLE OF CRYOPRESERVATION
 CRYOPROTECTANTS
 EXTENDERS
 DILUENTS

3.  CRYOPRESERVATION PROTOCOL
 FACTORS AFFECTING THE POST THAW
FERTILITY OF CRYOPRESERVED
SPERMATOZOA
 ADVANTAGES OF CRYOPRESERVATION
OF GAMETES

4.  Cryopreservation is the ex-situ conservation of
fish spermatozoa, eggs and embryos in ultra
low temperature such as -196°C
 cryopreserve the spermatozoa and ova from
various species and to use the same for seed
production
 Cryopreservation of female gametes of fish
could not be done successfully
 In india, the NBFGR is the primary
organization carrying out fish sperm
cryopreservation

5.  The feasibility of preservation of living cells in super
cold temperature
 Cryopreservation was first demonstrated by polge et
al in 1949
 This had stimulated extensive cryobiological
research
 J .h .s. blaxter first successfully cryopreserved the
spermatozoa of herring in 1953
 In india 27 species for sperm cryopreservation are
prioritized
 The NBFGR has initiated research programme on
cryopreservation of blastomeres of catfishes.

6.  Germplasm conservation
 Stock improvement for better growth
 Sex synchronization for research
 Desirable quality, quantity of gametes
 Available of disease resistance and pathogen free
seed
 sperm cryopreservation protocol can be an asset
where such milt related problem asynchronization
exsist
 The sperm cryopreservation has also been tested
for production of rohu seed

7.  Milt is the seminal fluid/seminal plasma
containing mature spermatozoa
 Seminal plasma is the source of nutrition for
the spermatozoa.
 Seminal plasma also inhibits sperm motility
 Motility of spermatozoa of salmonids last only
up to 2 minutes
 Once the fertility is lost, they loss there
fertilizing ability too
 Seminal plasma which is secreted by the
secretory cells lining

8.  Seminal plasma contains both organic and
inorganic components
 Monosaccharides and lipids are considered
essential for the nourishment of mature
spermatozoa
 Presence of K+ in the seminal plasma has been
shown to inhibit flagellar movement/motility
of the sperm
 Presence of citric acid in seminal plasma is
crucial to keep the sperm cells inactive in the
sperm duct
 In the seminal plasma, calcium and magnesium
are found bound to citric acid

9.  A few parameters have been developed to assess the
quality of sperms for cryopreservation
 Spermatocrit value
 Sperm density
 Milt volume
 Motility of spermatozoa
 Age of spermatozoa
 For the milt of carp, spermatocrit value of 70 and above
is considered good
 In case of indian major carps sperm density may vary
from 2×10⁷ to 3.5×10⁷ per cu.mm
 If percentage of spermatozoa exhibiting movement is
about 70% or more it is considered good
 For cryopreservation, milt from the middle of spawning
season has been shown to be good

10.  There are three essential steps in cryopreservation
 Freezing
 Storing
 Thawing
 The injuries to the cells may be due to
 Formation of ice crystals during freezing and thawing
 Due to osmotic changes
 The rate of freezing and rate of thawing are critical in
preserving the vitality and viability of the spermatozoa
 Moderate freezing is advisable
 If the cooling rate is too low, due to dehydration the cells die
 If the cooling rate is very high, the cells equilibrate
by intracellular freezing either by homogeneous heterogeneous
nucleation
 The formation of large intracellular crystal is fatal for the cells

12.  Vitrification is the process that transforms
intracellular water to noncrystalline solids after
freezing
 This occurs under two circumtances;
 If the cooling rate is very high, it does not allow
sufficient time for the water the water molecule to
crystallize
 The solution is so concentrated that the high
viscosity at low temperature does not allow water
molecule to get crystallize
 The temperature at which vitrification begins is
called glass transformation temperature
 Glass transformation temperature -13°C for water

13.  Cryoprotectants are chemicals that minimise cryoinjuries
to the cell due to ice formation or suppress ice formation
 The use of cryoprotectants increase the rate of
vitrification and optimise the post –thaw survival
 Cryoprotectants are of two categories
 Permeating cryoprotectants
 Non permeating cryoprotectants
PERMEATING CRYOPROTECTANTS
 Cryoprotectants that are permeable
to cell membrane
 Dimethyl sulfoxide (DMSO)m
 Glycerol,
 Methanol
 1,2 propanediol or propylene glycol

14.  It should be least toxic to the cells
 It should be permeable to the cells
 It should be soluble in water during freezing
 DMSO is the most commonly used cryoprotectants
 Methanol which is considered most toxic
 Glycerol is least toxic
 Though glycerol is least toxic, it is less permeable to
the cell membrane

15.  These chemicals are not permeable to cell membrane
 The commonly used cryoprotectant are;
 Sucrose
 Glucose polymers such as ;
Dextran
Hydroxyethylstarch
Poly vinyl pyrrolidone(PVP)
Egg yolk serum
Skim milk
Anti freeze protein
 They act by depressing the freezing point and raising the
glass transformation temperature of extracellular solution
 cryoprotectants plays the role of dehydrant, freezing point
depressant and extra/intra cellular stabilising agent

16.  It is a solution of balanced salts
 It is also inhibits the activation of spermatozoa
 This solution is meant to dilute milt as undiluted
milt is not suitable for freezing
 It functions as medium for cryoprotectant
 Usually, the diluents used for milt is the
combination of extenders and cryoprotectants
 For Indian major carps, 3 categories of extenders
have been found suitable

17. EXTENDER , A EXTENDER, B EXTENDER, C
Nacl 400mg 600mg 750mg
Cacl2 23mg 23mg 20mg
kcl 38mg 38mg 20mg
NaHco3 100mg 100mg 20mg
NaH2Po4 41mg 41mg
Mgso4 23mg 23mg
Dist.water 100mg 100mg 100mg

18.  Diluents is the combination of extenders and
cryoprotectants
 Three categories of diluents are prepared by mixing
cryoprotectants
ǀ ǁ ǁǀ
Extender, A -90ml Extender, B -90ml Extender, C -65ml
DMSO -5ml DMSO -8ml DMSO -15ml
Glycerol -10ml Glycerol -10ml
 Diluents can be mixed with milt at a proportion of 4:1 (
Diluents:Milt )

19.  It can be described in 5 phases
 Prefreezing
 Freezing
 Storage
 Thawing
 Post thaw and insemination
PRE-FREEZING PHASE
 Collection of milt from the milter
 Equilibration of milt diluents mixture
 The milt diluents mixture is kept at low
temperature for equilibration

21. FREEZING PHASE;
 The equilibrated milt –diluents mixture is
frozen at low temperature
 There are three ways of freezing viz;
 Pellet method
 It is extensively used for salmonids sperms
 Size of the pellet may vary from 50 to 200ml
 Straw method
 Straws are available in different volumes (0.25
to 4ml)
 This method is more popular during recent
years

22.  Ampoules/vials/ visitubes method
STORAGE PHASE;
 Frozen milt (pellet/ vial/ straw) is stored at
-196 °C
 Stored in liquid nitrogen, kept in cryocans
THAWING PHASE;
 For sperms frozen by pellet method, a thawing
solution is required
 One pellet may be placed in 1-2 ml of thawing
solution
 The straw are usually thawed in water bath at
30-40°C.

24.  Fast thawing is preferred as slow thawing may
cause crystallization
 In case of ampoules/ vials 65-70 seconds are
required for slush formation
POST THAW PHASE AND INSEMINATION;
 Sperm that have survived the cryopreservation are
ready for artificial insemination
 Artificial insemination is to be done immediately
after thawing
 Artificial insemination is to be done immediately
after thawing as there is reduction in the motility
period of spermatozoa after thawing

25. CRYOCANS

26. Phase Factors affecting
post thaw fertility
Prefreezing phase;  Quantity of donor males(age, size, health)
 Age of sperm and quality of milt
 Type of concentration of diluents
 Equlibration time and temp.
 Interval between sperm collecting and freezing
Freezing phase;  Freezing rate
 Cryoprotectant/ extender used
 Diluents preparation and dilution rate
 Type of freezing container(pellet/ straw/ ampoule)

27. Storage phase;  Temperature
 Back ground radiation
Thawing phase;  Thawing solution in case of pellets
 Thawing rate
 Motility induction and dilution
 Removal of cryoprotectants
Post thawing &
insemination;
 Sperm density for insemination
 Quality of donor female for eggs
Incubation

28.  It helps in the selective breeding
 It can help in preventing inbreeding depression
 It will be helpful in preserving genetic diversity
 This procedure shall be useful in case of fishes
where male and female mature at different
time
 It will be helpful in conserving endangered
species
 It can reduce the cost of male brood stock
maintenance

29.  Handbook of fisheries and aquaculture
Dr.S.Ayyappan
 Breeding of fin fish and shell fish
P.C.Thomas
 Applied fish genetics
B.K.Padhi
R.K.Mandal
 Google.nbfgr( encyclopedia )
 Google.wikipedia