CRYOPRESERVATION OF SELECTED SHELLFISH EMBRYOS AND EGGS Ta-Te Lin 1 and Nai-Hsien Chao 2 1....
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CRYOPRESERVATION OF SELECTED CRYOPRESERVATION OF SELECTED SHELLFISH EMBRYOS AND EGGS SHELLFISH EMBRYOS AND EGGS Ta-Te Lin 1 and Nai-Hsien Chao 2 1. Department of Agricultural Machinery Engineering, Nati onal Taiwan University, Taipei, Taiwan, R.O.C. 2. Department of Aquaculture, Taiwan Fishery Research Ins titute, Keelung, Taiwan, R.O.C.
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Transcript of CRYOPRESERVATION OF SELECTED SHELLFISH EMBRYOS AND EGGS Ta-Te Lin 1 and Nai-Hsien Chao 2 1....
CRYOPRESERVATION OF SELECTED CRYOPRESERVATION OF SELECTED SHELLFISH EMBRYOS AND EGGSSHELLFISH EMBRYOS AND EGGS
Ta-Te Lin1 and Nai-Hsien Chao2
1. Department of Agricultural Machinery Engineering, National Taiwan University, Taipei, Taiwan, R.O.C.
2. Department of Aquaculture, Taiwan Fishery Research Institute, Keelung, Taiwan, R.O.C.
INTRODUCTION
FUNDAMENTAL MEASUREMENTS
CRYOPRESERVATION PROTOCOLS
RESULTS
CONCLUSIONS
OSMOMETRIC CHARACTERISTRICSOSMOMETRIC CHARACTERISTRICS
EQUILIBRIUM OSMOMETRIC BEHAVIOR
NON-EQUILIBRIUM OSMOMETRIC BEHAVIOR
• HYDRAULIC CONDUCTIVITY
• CRYOPROTECTANT PERMEATION
MODELING AND PREDICTION
Comparisons of equilibrium osmometric behavior of oyster embryos, hard clam eggs and small abalone eggs.
1 / OSMOLALITY (osm-1)
0.5 1.5 2.50.0 1.0 2.0 3.0
FR
AC
TIO
NA
L V
OLU
ME
(V
/Vo)
0.2
0.4
0.6
0.8
1.2
1.4
1.6
1.8
2.2
0.0
1.0
2.0
OYSTER EMBRYOS, Vb = 0.49
HARD CLAM EGGS, Vb = 0.47
SMALL ABALONE EGGS, Vb = 0.45
Comparison of hydraulic conductivities for small abalone eggs and hard clam eggs.
1000/T (oK)
3.2 3.3 3.4 3.5 3.6 3.7 3.8
Lpx1
010
(m/s
-atm
)
100
101
102
103
HARD CLAM EGGS
SMALL ABALONE EGGS
30oC 20oC 10oC 0oC
Comparison of cell volume fluctuation in small abalone eggs during propylene glycol loading and unloading with or without osmotic buffer.
TIME (MIN)
0 10 20 30 40 50 60 70 80 90 100 110
FR
AC
TIO
NA
L V
OLU
ME
(V
/Vo)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
UNLOADINGLOADING
WITHOUT OSMOTIC BUFFER
WITH OSMOTIC BUFFER
TIME (MIN)
0 10 20 30 40 50 60 70 80 90 100 110
INT
RA
CE
LLU
LAR
CP
A C
ON
CE
NT
RA
TIO
N (
M)
0
1
2
3
4
5
6
7
UNLOADINGLOADING
WITHOUT OSMOTIC BUFFER
WITH OSMOTIC BUFFER
Comparison of intracellular CPA concentration in small abalone eggs during propylene glycol loading and unloading with or without osmotic buffer.
TOXICITY TOLERANCE TO CPATOXICITY TOLERANCE TO CPA
TIME
TEMPERATURE
CONCENTRATION
TYPES OF CPA
DEVELOPMENT STAGE
Toxicity tolerance of oyster embryos of various development stages to acetamide of various concentrations
6090
120150
180
210
240
1M2M
3M4M
5M
0
20
40
60
80
100
120
140
SURVIVAL (%)
DEVELOPMENT STAGE (MIN)
CONCENTRATION (M)
Toxicity tolerance of oyster embryos of various development stages to DMSO of various concentrations
6090
120150
180
210
240
1M2M
3M4M
5M
0
20
40
60
80
100
120
140
SURVIVAL (%)
DEVELOPMENT STAGE (MIN)
CONCENTRATION (M)
Toxicity tolerance of oyster embryos of various development stages to ethylene glycol (EG) of various concentrations
6090
120150
180
210
240
1M2M
3M4M
5M
0
20
40
60
80
100
120
140
SURVIVAL (%)
DEVELOPMENT STAGE (MIN)
CONCENTRATION (M)
CONCENTRATION (M)
Toxicity tolerance of oyster embryos of various development stages to propylene glycol (PG) of various concentrations
6090
120150
180
210
240
1M2M
3M4M
5M
0
20
40
60
80
100
120
140
SURVIVAL (%)
DEVELOPMENT STAGE (MIN)
INTRACELLULAR ICE FORMATION INTRACELLULAR ICE FORMATION CHARACTERISTRICSCHARACTERISTRICS
TEMPERATURE DEPENDENCY
TIME DEPENDENCY
EFFECT OF CPA
MODELING AND PREDICTION
CRYOPRESERVATION PROTOCOLCRYOPRESERVATION PROTOCOL
Two-step Freezing Procedure of Oyster and Hard Clam Two-step Freezing Procedure of Oyster and Hard Clam EmbryosEmbryos
4-Hr. oyster embryos cultured at 28oC
Loading with 2M DMSO, 10 min at 25oC
Cooling to -12oC at 1oC/min
Seedling at -12oC and holding for 5 min
Freezing to -35oC at 2oC/min
Holding at -35oC for 5 min
Quenching in liquid nitrogen
Storage
Rapid thawing in 28oC water bath
Experimental check point
Two-step Freezing Procedure of Oyster and Hard Clam Two-step Freezing Procedure of Oyster and Hard Clam EmbryosEmbryos
4-Hr. oyster embryos cultured at 28oC
Loading with 2M DMSO, 10 min at 25oC
Cooling to -12oC at 1oC/min
Seedling at -12oC and holding for 5 min
Freezing to -35oC at 4oC/min
Holding at -35oC for 5 min
Quenching in liquid nitrogen
Storage
Rapid thawing in 28oC water bath
Experimental check point
RESULTSRESULTS
TWO-STEP FREEZING PROTOCOLS OYSTER EMBRYOS AND EGGS SMALL ABALONE EMBRYOS HARD CLAM AND EGGS
VITRIFICATION OYSTER EMBRYOS SMALL ABALONE EMBRYOS
MODIFICATION OF THE TWO-STEP PROTOCOLMODIFICATION OF THE TWO-STEP PROTOCOL
Direct loading and unloading of 2M DMSO Optimizing seeding temperature Optimizing cooling rate Shortening holding time
Higher survival rate Immediate revival of oyster embryos with rotary motion
Survival of 4-Hr. oyster embryos at consecutive steps during freezing at a freezing rate of 2oC/min
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
LOADING
EFFECT
FREEZING
TO -35o
C
QUENCHINGIN LN2
ONE DAYIN LN2
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
LOADING
EFFECT
FREEZING
TO -35o
C
QUENCHINGIN LN2
ONE DAYIN LN2
Comparisons on the effect of adding trehalose to DMSO
LOADING
EFFECT
FREEZING
TO -35o
C
QUENCHINGIN LN2
ONE DAYIN LN2
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO 2M DMSO + 0.1M TREHALOSE
LOADING
EFFECT
FREEZING
TO -35o
C
QUENCHINGIN LN2
ONE DAYIN LN2
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO 2M DMSO + 0.1M TREHALOSE
Survival of 5-Hr. hard clam embryos at consecutive steps during freezing at a freezing rate of 2oC/min
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
LOADING
EFFECT
FREEZING
TO -35oC
QUENCHINGIN LN2
ONE DAYIN LN2
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
LOADING
EFFECT
FREEZING
TO -35oC
QUENCHINGIN LN2
ONE DAYIN LN2
Comparisons on the effect of using dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) as CPA.
LOADINGEFFECT
FREEZINGTO -35oC
QUENCHINGIN LN2
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO 2M EG 2M PG
LOADINGEFFECT
FREEZINGTO -35oC
QUENCHINGIN LN2
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO 2M EG 2M PG
Effect of freezing rate on the survival of cryopreserved oyster embryos loaded with 2M DMSO or 2M glycerol.
-2oC/min
FREEZING RATE
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO2M GLYCEROL
-4oC/min -16oC/min-1oC/min -2oC/min
FREEZING RATE
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO2M GLYCEROL
-4oC/min -16oC/min-1oC/min
Survival of Oyster Embryos at Consecutive Steps of VitrificSurvival of Oyster Embryos at Consecutive Steps of Vitrification Procedureation Procedure
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO 5M DMSO + 3M EG +
QUENCHINGIN LN2
6% PVP
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
2M DMSO 5M DMSO + 3M EG +
QUENCHINGIN LN2
6% PVP
Cooling rate = -0.5/min
EMBRYOS EGGSOYSTEROYSTERCONTROL
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
110
COOLING RATE = -0.5oC/minCooling rate = -0.5/min
EMBRYOS EGGSOYSTEROYSTERCONTROL
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%
)
0
10
20
30
40
50
60
70
80
90
100
110
COOLING RATE = -0.5oC/min
Survival rates of cryopreserved oyster embryos and eggs
Quenching in LN2
-16oC-14oC-10oC -12oCSEEDING TEMPERATURE (oC)
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%)
0
10
20
30
40
50
60
70
80
90
100
QUENCHING IN LN2
FREEZING TO -35oC Quenching in LN2
-16oC-14oC-10oC -12oCSEEDING TEMPERATURE (oC)
SU
RV
IVA
L O
VE
R C
ON
TR
OL
(%)
0
10
20
30
40
50
60
70
80
90
100
QUENCHING IN LN2
FREEZING TO -35oC
Effect of seeding temperature on the survival of cryopreserved oyster embryos
Schematic of Conventional Two-Step Cryopreservation Procedure
Temperature(oC)
Time
0
Equilibrate in Cryoprotectant
SeedingStart slow cooling
Hold for equilibrationQuenching in LN2
-196
SUMMARYSUMMARY
Oyster embryos(Crassostrea gigas)
Hard clam embryos(Meretrix lusoria)
Small abalone embryos(Haliotis diversicolor diversicolor)
SHELLFISH
METHODTwo-step freezing Vitrification
788%
7210% Unknown
148%
< 1%Negative
CONCLUSIONSCONCLUSIONS
Fundamental measurement of osmometric, intracellular ice formation characteristics and toxicity tolerance helps in designing and optimizing the cryopreservation procedures for selected shellfish embryos and eggs.
A 788% and 7210% survival rate over control for oyster and hard clam embryos were achieved after optimization of the freezing procedure.
Survival of small abalone and oyster embryos with the current vitrification procedure was <1% and 148%, respectively. Both required further optimization of the vitrification procedure.
