CRYOPRESERVATION AND its

Application in aquaculture
AND HUMAN WELFARE

Prof. N. Munuswamy,
Munuswamy, D. Sc.,

Department of Zoology
University of Madras
Guindy Campus
Chennai – 600 025

Endowment lecture, Dept. of Zoology, Annamalai University 18th March 2011.

 Role of water in cell survival
intracellular water 60 - 90%

Retain Removal 0°C

intra cellular ice extra cellular ice

-10 °C
ice crystal growth

shrinkage
cell death
cryopreserve / thaw
survival
 ANTIFREEZE PROTEINS
What is an antifreeze protein?
• AFP is a protein with affinity for ice.

Many animals that live in extremely cold

climates produce proteins that act to prevent the
formation of ice crystals in the body fluids of the
organism. Antarctic fish produce a range of such
proteins, as the water temperatures can drop below 0 o
C. We have determined the structure of the type I
antifreeze protein (SS3) from the blood of the shorthorn
sculpin at both 5 degrees and -5 degrees. The structure
largely comprises a long alpha-helix that displays
conserved Thr residues (red) along a single surface.
These residues are important for preventing the growth
of ice crystals, although the mechanism of action is not
currently understood. An additional short helix exists at
the N-terminus of the protein, although the
conformational relationship between the two helices is
not well defined. This is the first solution structure of a
wild-type type I antifreeze protein
AFP ACTIVITIES

Animals that rely on freeze resistance for survival (like

some fishes and insects) often use antifreeze proteins to
depress their freezing point below subzero ambient
temperature.

Other organisms (like plants and soil bacteria) that

tolerate freezing can use antifreeze proteins to inhibit
recrystallization of ice, ie. stop the growth of large crystals
at the expense of small ones.

Fig. 2. Recrystallization inhibition. Here are two frozen

solutions viewed under a microscope in a time-lapse
sequence. The ice in the upper panel contains AFP, which
keeps the crystals tiny. The lower panel lacks AFP. The
number of crystals goes down as their size increases.
How do AFPs bind to ice?

The binding of AFPs to ice can be considered as a receptor:ligand interaction. It

was solved by investigating the three-dimensional structure of the AFP using NMR
or X-ray crystallography; identifying the ice-binding surface by site-directed
mutagenesis, determining which surface of ice is bound by the AFP using ice
etching, and then integrating this information by modelling the docking of the AFP to
ice.

Model of shorthorn sculpin (fish) AFP binding to a secondary prism plane of ice.
.
Ref: Baardsnes et al., “Antifreeze Protein from Shorthorn Sculpin: Identification of the Ice-Binding Surface”. (2001) Protein Science 10, 2566-2576
 Mechanism of Antifreezing Activity

α-axis

Hydrogen Oxygen

Fig.2A. Model of flounder antifreeze hydrogens Fig.2B. Antifreeze molecules adsorbed on the
bonded to prism face of hexagonal ice, parallel to basal plane interfere with the propagation of
one of the α-axes. steps across the basal plane because the steps
become highly curved.
 AFP diversity

There are five very different types of AFP in fishes. In some cases
their molecular ancestors have been identified. Thus the type II AFPs
are clearly derived from C-type lectins. Their diversity may reflect
recent evolution and the ability of ice to present many different
surfaces for binding.
 Illustration of hydrogen-bonding hypothesis for AFP binding to ice

Thr and Asx side chains of AFP hydrogen bonding to oxygen atoms on the prism plane of ice
(De Vries, 1983)
Antifreeze significantly increases the AFPs inhibit ice recrystallization at low
plasma freezing point of Atlantic concentrations. Crystals measurement
fishes. Comparison of the freezing point across their estimated maximum dimension
of seawater (approx. -1.8 °C) with the during an 18-h period after freezing with no
plasma freezing points of six teleost antifreeze (control) or in the presence of 10-6
species of Atlantic Canada. Plasma- or
freezing points closely approximate to 10-4 mg/mL AFGP.
the whole fish freezing point or lethal
temperature.
Thatched - bars represent enhancement
of freeze protection due to antifreeze
production.
A selection of studies that used AFP or AFGP to improve the survival of various cells
or tissues

Storage
Cell or Tissue Antifreeze Type
tempa
Reference

Fish sperm b AFGP C 64

Carp sperm AFGP C 65

Mouse embryous AFGP C 66

Mouse oocytes AFGP C 67

Rat liver AFP Type III H 57

Ram sperm AFP Type I, AFGP C 68

Pig oocytes AFP Type I, II, and III; AFGP C 69

Bovine oocytes AFP Type I, II, and III H 55

Chimpanzee sperm AFP Type III C 70

Human oocytes AFP Type I, III H 71

Human platelets AFGP H 63

a H, hypothermic; C, cryogenic.
b Rainbow trout, steelhead salmon, carp.
Oocytes incubated in AFP solution survive
cold storage. Bovine oocytes were incubated
for 24 h at 4 °C in the absence (control) or
presence of AFP Type I, II, III, or antifreeze
glycoprotein (AFGP) from various marine
teleosts.
AFGPs protect cold-stored human blood platelets. Blood platelets were stored at 4 °C
without (control) or with AFGP. More than 90% of the control platelets self-activated within
15 days. The majority of treated platelets did not self-activate during 21 days of storage.
Throughout this period, platelets appeared to be capable of activation as evidenced by their
response to thrombin (vertical arrows).
Potential use of AFPs to preserve the texture of frozen meats. Scanning
electron micrographs of bovine muscle tissue frozen in the absence of AFGP
(A) and frozen after soaking in an antifreeze-containing solution (B).
Without AFGP, large intracellular ice crystals formed throughout the tissue,
leaving cavities on thawing. Scale bar = 50 µm.
 Winter flounder Salmon

Incorporation of AFP gene (Flounder AFP gene) into

salmon gene (Salmon eggs) to produce transgenic
Salmon

1986-1988
Observations to check the incorporation of AFP transgene into host
genome by Southern Blot transfer
 Transgenic flounder males crossed with wild type female

1988-1989

F1 Screened for transgenes (33-64%)

F1 Transgenic males crossed with wild female

1990
F2 Screened for transgenes (51-54%)

F1 Trangenics produce Pro-AFP

(0.07-20 ng/ml)
 Future Direction
• Increase Initial gene (AFP) dosage
• Designing better AFPs
• Increase gene dosage through cross breeding
PRESERVATION STUDIES IN FINFISH
GAMETES
 TYPES OF CRYOPROTECTANTS
PERMEATING CRYOPROTECTANTS

Dimethyl sulphoxide (DMSO)

Ethylene glycol

Propylene glycol

Methanol

Glycerol (natural CPA)
TOXICITY NEUTRALIZERS
 Acetamide
 Formamide
NON-PERMEATING CRYOPROTECTANTS
 Polyvinyl pyrrolidone (Mw 40000)
 Trehalose
NATURALLY OCCURRING CRYOPROTECTANTS

Anti-freeze protein

Antihysterisis protein
CHARACTERISTICS OF CRYOPROTECTANTS

Non-toxic or less toxic

Non – electrolyte and serve as salt buffers

Easily permeable

Freezing point - depressant

Easily miscible in water

 Sea bass Lates calcarifer
 Economically important, popular edible species,
commanding premium price in market
 Euryhaline and catadromous
 Distributed in tropical and sub-tropical areas of the
Western-Pacific and Indian ocean

 Milt: Seminal plasma bearing spermatozoa

 Extender : Medium which avoids the activation of
spermatozoa during cryopreservation (Graybill and
Horton, 1969)
 Short-term, refrigerated storage of milt:
Developed for several teleosts, such as
walleye, red drum, Atlantic sturgeon, milkfish,
Mozambique tilapia, paddlefish, channel
catfish, salmonids and striped bass
 Assessment of osmolality

Samples Osmolality (mOsm kg-1)

Seminal plasma 341±4

Modified fish ringer solution 337±3.3

Х ± SD of 3 replicates
 Motility of spermatozoa of L. calcarifer at two hours of
successive time intervals in modified fish ringer solution

Motility (%)
Dilution
(Milt:Extender)
2nd h 4th h 6th h 8th h

1:10 83±1.25 78±0.95 71±1.5 60±2.06

1:20 87±0.81 84±2.08 79±1.29 71±1.25

1:30 78±0.95 71±1.29 62±1.91 50±1.29

1:40 72±1.25 63±1.73 51±1.5 36±1.29

Х ± SD of 3 replicates
 Photomicrographs showing spermatozoa of Lates
calcarifer at 1:20 (milt:extender) dilution ratio during
selected hours of preservation

2nd hr 4th hr

8th hr
6th hr
Protein, lipid and carbohydrate content of seminal plasma of
L. calcarifer in various dilution ratios in the extender

Dilution ratio Protein Lipid Carbohydrate

(mg/ml) (µg/µl) (µg/µl)

Control 0.76 0.22 0.11

1:10 0.80 0.25 0.13
1:20 0.78 0.24 0.12
1:30 0.85 0.28 0.16
1:40 0.92 0.32 0.18

Х ± SD of 3 replicates
 PANGASIUS SUTCHI

Pangasiid catfish living

in the lower part of the
Mekong River (Sauvage,
1880 in Roberts and
Vidthayanon, 1991).

This species is widely

exploited for cage culture
in the Mekong Delta
(Cacot, 1993).
Protocol for sample preparation for toxicity assay

Collection of Milt sample in a cryovial

Addition of selected Cryoprotectants (5%/ 10%/ 15%/ 20%)

Equilibration for 20 min at RT

Gradual dilution of the medium with suitable extender (HBSS)

Stored as different temperature after equilibration time

Estimation of viability based on dye exclusion technique

 STRIPPING

Anesthetized with ms222 Stripping of milt from Pangasius sutchi

 Cryoprotectant Toxicity assay

100
90
80
70
60
% Motility

50
40
30
20
10
0

Cryoprotectants
 CRYOPROTECTANT TOXICITY ASSAY

A B
sp
sp

C D

SZ
sp

DMSO 15% 83.33

83.33±
±1.24
 Assessment of sperm morphology

 Note the viable, round shape

spermatozoa, maintained in extender
(HBSS) mixed with filtered fresh water.
x 40.

SZ
 Viability of sperm under experimental conditions

 Viability assessment using eosin

and nigrosin stain.

 Note:Viable sperm excludes stain

and dead sperms takes up stain x
100
D

sp

L
 Percent motility of spermatozoa of Pangasius sutchi at
different dilution ratio of cryoprotectants
100

90

80

70

60
Methanol
DMSO
% Motility

50
propane diol
Ethane diol
40 Dimethyl acetamide

30

20

10

0
1:10 1:20 1:30 1:40

different dilution ratio

Cryopreservation protocol for
crustacean gametes and larvae
 Cryopreservation protocol for gametes and larvae

Sample collection

Testing of cryoprotectant toxicity

Selection of extender or diluent media (maintenance of
morphological integrity over extended period of time at
physiological temperatures)

Addition of cryoprotectant at lower temperatures (- 15oC) is less
cryotoxic

Rate of freezing in programmable freezer (range 0. 5oC/ min. to -15oC/ min.)

Seeding – to ensure uniform ice crystal formation in external medium

Final cryogenic storing in liquid nitrogen at -196oC

Thawing – rapid thawing at 75oC, slow thawing by immersing in water bath
at room temperature or in air

Dilution - Dropwise addition of cryoprotectant-free extender OR stepwise
dilution with addition of progressively lower concentration of cryoprotectants
and final transfer to extender

Testing the viability
 Equipments purchased – DST Project
Leica DM 2500 Trinocular phase contrast microscope with DIC

Specification
 Brightfield
 Darkfield
 Phase contrast
 Polarization
 Differential Interference Contrast
 Cryotolerance objectives (20X)

Cryostorage Containers “INOX”,

Quantity--3 nos.
Quantity

3.9 lt

10.5 lt

20.5 lt
CRYOMICROSCOPE PROGRAMMABLE FREEZER
Types of Freezing and their mechanism in Shrimp larva
Room temp. RT or less -7oC Seeding -30oC -196oC
(RT) or less
Slow Freeze

- 2.5oC/ min. - 2.5oC/ min. Plunge

Equlibration -1500oC/
min.
Cryoprotectant Medium and larva Dehydrated and Partially frozen
permeated larva supercooled partially frozen larva and vitrified larva
RT or less RT or less o
-7 C Seeding -30oC
Rapid Freezing

- 2.5oC/ min. - 5oC/ min. Plunge

Equlibration -1500oC/
min.

Cryoprotectant Medium and larva Dehydrated and Partially frozen

permeated larva supercooled partially frozen larva and vitrified larva
RT or less RT or less -196oC Vitrified medium
Vitrification

Plunge
Supercooled medium

Equlibration > -1500oC/ Partially Frozen medium

min.
Dehydrated larva Fully vitrified Partially Frozen and
medium and larva Vitrified medium
 HISTORICAL OVERVIEW OF MALE GAMETE CRYOPRESERVATION

Extender/
Species/ Author Temp. Duration Viability Survival
CPA
M. rosenbergii – Ringer 2o C 7 days Al-Fert. Hatching
Chow 1982 solution

M. rosenbergii – G in FW -196oC Upto 30 Al. Fert Hatch/

Chow et al. 1982 days Larval
metamorph
Joshi and Diwan Ringer 6o C 96 hrs. Fert. Hatching

H. americanus – Paraffin oil 4 - 7oC Upto 215 Morphol./ ~ 55%

Ishida et al. 1985 days AR
A23187

S. ingentis – Suc, Trehal, -196oC 1 month AR egg 37%

Anchrodoguy et DMSO, water
al. 1988 Prol., G in
ASW
Species/ Author Extender/ Temp. Duration Viability Survival
CPA

P. indicus – G, Trehal - 196oC 1 week AR 80 %

Diwan et al. - 35oC 1 week AR 76%
1994

L. vannamei – SW/ 15oC 36 hrs. Morphol. ~ 82%

Bray and Ca++FASW
Lawrence 1998

S. Serrata – G, DMSO, - 196oC 1 month Dye 95%

Jeyalectumie Trehal, exclusion
and NaCl+ PBS
Subramoniam
1989
Species/ Author Extender/ Temp. Duration Viability Survival
CPA

S. Serrata – G, M, EG, -196oC 30 days 57-72 %

Bhavanishankar DMSO in 2-4oC 72 hrs. 82%
1996 Ca++FAS -196oC 30 days Dye 39-42%
W -196oC 30 days exclusion 37-41%
-196oC 30 days 8%
S. Serrata – G, M, EG, -196oC 8hr./ 30 HOST, Dye
Bhavanishankar exclusion, 52%
DMSO in days
and AR A23187
Ca++FAS
Subramoniam
1997 W

30 days
Rotifer M + DMSO -196oC Ice crystal -Cryo injury
formation
Munuswamy
2007
 Viability assessment in cryopreserved sperm
Indices for testing post-thaw viability
•Staining techniques
•Osmotic sensitivity tests
•Induced acrosome reaction
•Fertility
sperm suspension + 0.5% eosin followed by 10% nigrosin or trypan blue
(1%) + sperm suspension. Dye exclusion , taken as index for survival.

Sperm released in hypo-/hyper-osmotic media. (2:1 mixture of

Ca++FASW and DDH2O/ 5-10% sucrose in Ca++FASW) - osmotic
response taken as index for membrane integrity.

Sperm incubated in CaCl2 in Ca++FASW; subjected to ionophore

treatment (A23187); incubated and scored for AR/Sperm suspension
incubated in SW containing egg derivatives and scored for AR AI and
subsequent hatchability
Cryopreservation of spermatozoa of mud crab
Scylla serrata
 Acrosome reaction in the spermatozoa of Scylla serrata

1 Normal spermatozoon. The reaction is initiated with retraction of nuclear arms

2–3 and swelling of nuclear mass
3–4, followed by eversion of the subacrosomal material through the apical
acrosomal cap
5–6 (arrows in 6 indicate the direction of progressive eversion) and culminates in
the formation of acrosomal filament (7).
Bar = 4 µm.
 Freezing damage in the spermatozoa of Scylla serrata.

1. Normal spermatozoon
2–6. Morphological damage in the regions of
nucleus and acrosome due to injuries during freezing. Bar = 4 mm.
Cryopreservation of nauplii and embryo of Indian
white shrimp
Fenneropenaeus indicus
 Crustacean embryo cryopreservation

Limitations

embryonic stages:
 presence of yolk, hatching envelope and embryonic
envelope affect CPA transport
 information on CPA permeability
 sensitivity to chilling and freezing

larval stages:
 chitinous body wall/basement membranes
 organogenesis/structural complexity
 several barriers limit total CPA permeation
 susceptibility to intracellular ice formation
 FREEZING OF PRAWN NAUPLII
EXPERIMENTAL PROTOCOL
Cryoprotectant Ethylene glycol, 15% v/v
Addition Two steps at 15oC
Equilibration time 15 min.
Cooling rate - 1.5oC/ min.
Seeding - 6 oC
Final temperature - 40oC
Thawing rate Rapid > 300oC/ min.
Dilution Slow
Stage VI nauplius of P. indicus frozen Eversion (arrows) in vitrified stage II
to -40oC using the slow cooling nauplius of P. indicus when the
procedure and thawed at > 300oC/ vitrification medium was diluted
min..
min step--wise
step
Freezing of nauplii of P. monodon frozen in 33 ppt sea water
in 20% v/v EG

-15.4°C -19.0°C -22.3°C Photographic

reproduction of
videomicrographed
images
A B C
Cryomicrographic
-6.8°C -0.7°C 2.7°C observations made on
Planer CM3 cryostage under
bright field, using Zeiss
Axioplan Universal
Microscope
D E F
15.0°C
Freezing/thawing velocities (2°
(2°C/min)
Note growth of ice in B. Flashing occurred in
sample indicative of intracellular freezing
G
 Freezing/Fracture injury in nauplii and
protozoeae of P. indicus
A C E

B D F

Fig A: arrows indicate irregularities in CPA exit; Fig. B: notice body-wall

leakage; Fig. C: dissociation of internal tissues in the head region;
Fig. D: damage to alimentary tract; Fig. E & F: Fracture injury in vitrified
nauplii
 Freezing injury in early embryonic stages of
P. indicus
A C
Fig A & B: Notice individual
blastomeres

139 µm

Fig. C&D: dissociation of internal

B D
cell mass and dislocation of
blastomeres below the embryonic
envelopes (blastula stage)
CRYOPRESERVATION OF SHELLFISH GAMETES
(Macrobrachium rosenbergii)
 Aquaculture species
 Only species of the genus Macrobrachium have been seriously considered
for aquaculture
 150 species of Macrobrachium are distributed throughout the tropical and
subtropical regions of the world.
world.
 They occur in most inland freshwater areas such as lakes, rivers, swamps,
estuaries
 Many require brackish water for their early development, although others
can complete their life cycle in fresh water
 Cryopreservation protocol for gametes

Electroejaculation of spermatophores

Addition of cryoprotectants (5%/ 10%/ 15%/ 20%)

Equilibration for 15 min

Selection of a suitable extender

Stored as different temperature after equilibration time

Formulation of a suitable protocol for long term preservation in LN2(-196º)

Estimation of viability based on dye exclusion technique, acrosome reaction &

artificial insemination
 Collection of spermatophores

• Electroejaculation

SP

S P - Spermatophore
 Extenders

Cortland medium Fish ringer solution

(284 mOsm±1.2) (278 m Osm ±2.4)

Viability % (34 ±1.37) Viability % (45 ± 2.3)

Hanks Balanced salt solution Filtered Pond water

(292 mOsm ±0.05) 120 mOsm ±0.02

Viability % (65 ± 0.09) Viability % (75 ±1.5)

 Toxicity assay
Viability %

100
80
60
40
20
0
 Viability of spermatophores
(Dye exclusion principle)

DS

LS
LS
DS

Eosin Nigrosin Trypan Blue

Note: Viable sperm excludes stain and dead sperms takes up stain x 20
Short term Preservation of spermatophores at 4°C & -20°C
for about 5,10 ,15 & 20 days

S.No Storage at % viability Storage at - % viability

4ºC 20ºC

1 5 days 74±0.23 5days 77±2.12

2 10 days 66±0.12 10 days 65±3.61

3 15 days 58±0.76 15 days 53±0.12

4 20 days 56±1.87 20 days 51±1.32

 Acrosome reaction in sperm of freshwater prawn
Macrobrachium rosenbergii

A B C

F
D E
 SEM of mature sperm of M.rosenbergi

MS

A B
MS

MS

c D
MS – Mature sperm
 Further studies

• DNA damage studies • Flow cytometry

• Fluorescence microscopy
DNA damage studies of cryopreserved gametes
Using Comet assay technique

A
 Flow cytometric evaluation

Micrographs of bovine (a), porcine (b), canine (c), equine (d), murine (e), and human (f) spermatozoa that had been stained sequentially with
carboxyfluorescein diacetate (CFDA) and propidium iodide (P1) and viewed with an epifluorescence microscope (X 800). Spermatozoa
(Garner et al .,1986).
Human sperm stained with the Molecular Probes Live/Dead Sperm
Viability Kit showing green (live) and red (dead) sperm under
fluorescence microscopy (Eshan Senanayake et al
al..,2010)
2010)
 CRYOBIOLOGY OF ROTIFERS
(Brachionus calyciflorus & B. Plicatilis)
B. calyciflorus B. plicatilis

(Freshwater) (Marine)
Developmental stages of rotifers
 Developmental stages of rotifer B. plicatilis and B. calyciflorus

Age of embryo (hrs.)

Expected stages
B. plicatilis B. calyciflorus

I 2-3 2-3
II 3-5 3-6
III 4-7 6-9
IV 4-8 9-12
Hatch 8-11 12-15

B. plicatilis B. calyciflorus

IV stage corresponding to eye-stage was found to be suitable for

cryopreservation as it showed maximum survival after thawing. This
stage of development seems to be amenable for cryopreservation of
rotifers embryo
 B. plicatilis B. calyciflorus
Note: The stained animal and the unstained Embryo
 Permeability in the embryo was comparatively negligible as
evident by the non-entry of the stain.

 It elucidates that the egg membrane is impermeable to water

when subjected to isotonic solution. When exposed to the
stain for a period of ten minutes only adult rotifers stained,
where as the embryo did not takeup the stain

 Thereafter, rotifer was transferred to the fresh medium; the

rotifer adult immediately lost the stain with the influx of fresh
medium into its body. This process is evident in both rotifer (B.
plicatilis and B. calyciflorus)
a b

Note viable rotifers subjected to DMSO (10%) showing accumulation of

cryoprotectant. a). B. calyciflorus embryo b). B. plicatilis adult.
c) B. calyciflorus adult
 Rotifer B. plicatilis exposure to cryoprotectant before frozen (10% DMSO)

Post thaw survival of B. plicatilis with out removal of cryoprotectant (10% DMSO)

Accumulation of
DMSO
 Percent post-thaw survival of B. plicatilis (at the rate of – 10C/min and - 2.5 0C/min
for embryos and -200C/min and -300C/min for adults)

60
-1
-2.5
-20
-30
50

40
Survival (%)

30

20

10

0
Methanol 1,2 Ethanediol DMSO 1,2 Propanediol
Cryoprotectents
cryoprotectants

-10C / min -200C / min

Embryo Adult
-2.50C / min -30C / min
 Percent post-thaw survival of B. calyciflorus (at the rate of
0-10/min and -2.5 0C/min for embryos and -200C/min and -300C/min for adults)

70

-1
-2.5
60 -20
-30

50

40
Survival (%)

30

20

10

0
Methanol 1,2 Ethanediol DMSO 1,2 Propanediol
Cryoprotections
Cryoprotectants

-10C / min -200C / min

Embryo Adult
-2.50C / min -30C / min
 Freezing Methodology

Semi-Controlled Rate Controlled

Freezing rate(1°C – 25 ° C/min.) Freezing rate(0.1 ° C – 30 °

C/min.)

Samples in CPA Samples in CPA

Liquid Nitrogen Freeze Program(Programmable freezer)

Thermal Calibration Plunged into Liquid Nitrogen

(Platinum Resistance Thermometer or
Alcohol Thermometer)
Sequence of freezing - experimental setup
programming
Loading LN2 and connecting to Planner
Sample preparation in vials and Straw
Straw with sample is being attach to sample holder

sample holder transfer to cryo chamber

Remove the sample from cryo-
cryo-chamber Plunging the sample after run the programme
Removing the sample for viability assay Storage container used for
storing the cryopreserved sample
Training programme on cryopreservation…….
 Thaw Procedure

Initial Thawing (37 ° C – 15sec in water bath)

Final thawing (24 ° C)

Assessment for Viability

Adult Embryo
(Rhodamine test) (Hatching test )
Permeability assessment using 0.1% Rhodamine B

Permeability Time taken for Time taken for the

entry of stain (min) stain to leave the
system completely
(min)

Adults +++++ 5 20

Embryo - - -

Note: The stained animal and the unstained Embryo

Sequence of freeze events in B. calyciflorus- Freshwater medium

e - Freezing around the rotifer

e-i - Blackening of rotifer as a result of ice formation within the body
k - Rotifers loose their opacity on thawing
Sequence of freeze events in B. calyciflorus – 10% DMSO

b - Pattern of ice crystal growth

e-g - Blackening of rotifer as a result of ice formation within the body
h,i - Rotifers after thawing to room temperature
Sequence of freeze events in B. calyciflorus suspended in 15 %
methanol subjected to - 30°C. Here the medium freezes completely
but the rotifer did not freeze until -30°C. (Bar – 100 µm)
 Post thaw survival of rotifer B. plicatilis
(after removal of cryoprotectant)

Period of preservation: 30 days (LN2)

Post thaw survival of rotifer B. plicatilis

Cryoprotectants Survival (%)

DMSO 43.30 ± 0.57

1-2 Propanediol 26. 06 ±1.15

MeOH 48.40 ± 3.15

Glycerol 13.00 ± 3..32

Ethylene Glycol 18.6 ± 2.70

Scanning electron micrographs show the rotifer treated with 10% MeOH
after post thaw embryo

A B

C
D
A- Normal parthenogenetic egg
B,C& D - treated with cryoprotectant (MeOH)
 Scanning electron micrographs show the rotifer treated
with 10% DMSO after post thaw embryo

A B

C D

Note: Damages on the egg surface of the

embryo (B. plicatilis) during cryo injury
Slow freezing rate injuries
Scanning electron micrographs show the rotifer treated with 10% DMSO
after post thaw embryo

A B

Rapid freezing damage

 Expression of HSP70 as biomarker in the rotifer embryos
exposed to various cryoprotectant

Lane 1. Propanediol
Lane 2. Ethylene glycol
Lane 3. Glycerol
Lane 4. Dimethyl sulphoxide
Lane 5. Methanol
Lane 6. Normal rotifer (without cryoprotectant)
Note the occurrence of HSP 70 in rotifer subjected to
various cryoprotectants

Western blot analysis of rotifers supernatant to HSP 70 detected a single clear

prominent band with an estimated molecular weight of ~70 kDa (Lane 1-6) and
molecular mass standard β–actin used as an internal control

Bar diagrams represent level of HSP 70 expression in cryoprotectants treated groups

compared with β–actin expression as internal control.
 RT-PCR pattern showing the HSP 70 expression of rotifers treated with
10% concentration of cryoprotectants with β–actin as internal control.
The expected amplican size of 579bp of gene in rotifers subjected to
cryoprotectants

bp . Ladder 1000 bp
Lane 1. Dimethyl sulphoxide
Lane 2. Ethylene glycol
Lane 3. Glycerol
Lane 4. Methanol
Lane 5. Propanediol
Lane 6. Normal rotifer (without cryoprotectant)
CRYOPRESERVATION OF HUMAN
GAMETES & EMBRYOS
 SPERM PRESERVATION PROTOCOL
SPERM FREEZE SOLUTION ( 0.7 ml) + SEMEN (1 ml)
(15% Glycerol + 0.4% Human serum
albumin in HEPES buffered solution)
THAWING CRYOPRESERVATION

ESTIMATE MOTILITY (Pre-

(Pre-freeze motility)

ALIQUOT TO CRYOVIAL

ALLOW TUBES TO STAND IN LIQUID NITROGEN

VAPOUR FOR 15 min.

PLUNGING IN LIQUID NITROGEN

REMOVE TUBES FROM LIQUID NITROGEN AND

WARM AT ROOM TEMPERATURE FOR 30 min.

ASSESS POST-
POST-THAW MOTILITY
HUMAN SPERMATOZOA
 Cryopreservation of Human embryos
For early cleaving embryos
Embryos incubated in 1.5M 1,2-
1,2-propanediol for 10 min. at room
temperature

Embryos transferred to 1.5M 1,2-
1,2-propanediol + 0.1M Sucrose
FREEZING


Loaded into straws

Straws loaded into programmable freezer

Freezing begins at 20oC and brought down to -150oC

Straws transferred to liquid nitrogen

Straws are thawed in air for 30 sec and then in 30oC water bath for 40 sec

Embryos released into 1M 1,2-
1,2-propanediol with 0.2M sucrose
THAWING


Embryos transferred to 0.5M 1,2-
1,2-propanediol with 0.2M sucrose

Embryos transferred to 0.2M sucrose

Embryos transferred to culture medium
 Cryopreservation of Human embryos
For blastocysts
Blastocysts incubated in 5% glycerol

Transferred to 10% glycerol + 0.1M Sucrose

FREEZING

Loaded into straws


Straws loaded into programmable freezer

Freezing begins at 20oC and brought down to -150oC

Straws transferred to liquid nitrogen

Straws are thawed in air for 30 sec and then in 30oC water bath for 40 sec

Blastocysts released into 10% glycerol with 0.2M sucrose
THAWING


Transferred to 5 glycerol with 0.2M sucrose

Blastocysts transferred to 0.2M sucrose

Transferred to culture medium

Observation to see whether blastocoel has expanded
Mature oocyte Intracytoplasmic sperm injection

Fertilized egg
 Human Embryos

2 celled stage 4 celled stage

8 celled stage
Blastocyst Blastocyst hatching
Blastocyst shrinking during Post thaw blastocyst fully
freezing expanded
 APPLICATION OF CRYOPRESERVATION
IN AQUATIC SCIENCES
Hybridization between species with temporal spawning differences

Ameliorate seed scarcity

Maintenance of desired gremplasm free of genetic contamination
for stock improvement
Maintenance of desired somatic cell lines/ tissues for experimental use

Transportation of seed/ tissues/ cells etc.

Artificial insemination
In vitro fertilization
Conservation of genetic strains of commercially important
and endangered species
Tasks accomplished
 Spermatozoa preservation in Scylla serrata
 Nauplii and embryo preservation in Fenneropenaeus indicus
 Short
Short--term, refrigerated storage of milt Sea bass Lates
calcarifer
 Cryopreservation of rotifer B. calyciflorus using high and
low molecular weight cryoprotectants
My sincere thanks to.......................

• The authority of Annamalai University

• Prof. Selvi Sabhanayagam &
• Faculty members of Zoology
Department, Annamalai Unievrsity