CRYOPRESERVATION OF SELECTED SHELLFISH EMBRYOS AND EGGS TaTe

CRYOPRESERVATION OF SELECTED SHELLFISH EMBRYOS AND EGGS Ta-Te Lin 1 and Nai-Hsien Chao 2 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.

l INTRODUCTION l FUNDAMENTAL MEASUREMENTS l CRYOPRESERVATION PROTOCOLS l RESULTS l CONCLUSIONS

OSMOMETRIC CHARACTERISTRICS u EQUILIBRIUM OSMOMETRIC BEHAVIOR u NON-EQUILIBRIUM OSMOMETRIC BEHAV • HYDRAULIC CONDUCTIVITY • CRYOPROTECTANT PERMEATION u MODELING AND PREDICTION

Comparisons of equilibrium osmometric behavior of oyster embryos, hard clam eggs and small abalone eggs.

Comparison of hydraulic conductivities for small abalone eggs and hard clam eggs.

Comparison of cell volume fluctuation in small abalone eggs during propylene glycol loading and unloading with or without 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 CPA u TIME u TEMPERATURE u CONCENTRATION u TYPES OF CPA u DEVELOPMENT STAGE

Toxicity tolerance of oyster embryos of various development stages to acetamide of various concentrations

Toxicity tolerance of oyster embryos of various development stages to DMSO of various concentrations CONCENTRATION (M)

Toxicity tolerance of oyster embryos of various development stages to ethylene glycol (EG) of various concentrations CONCENTRATION (M)

Toxicity tolerance of oyster embryos of various development stages to propylene glycol (PG) of various concentrations CONCENTRATION (M)

INTRACELLULAR ICE FORMATION CHARACTERISTRICS u TEMPERATURE DEPENDENCY u TIME DEPENDENCY u EFFECT OF CPA u MODELING AND PREDICTION

CRYOPRESERVATION PROTOCOL

Two-step Freezing Procedure of Oyster and Hard Clam Embryos 4 -Hr. oyster embryos cultured at 28 o. C Loading with 2 M DMSO, 10 min at 25 o. C Cooling to -12 o. C at 1 o. C/min Seedling at -12 o. C and holding for 5 min Freezing to -35 o. C at 2 o. C/min Holding at -35 o. C for 5 min Quenching in liquid nitrogen Storage Rapid thawing in 28 o. C water bath Experimental check point

Two-step Freezing Procedure of Oyster and Hard Clam Embryos 4 -Hr. oyster embryos cultured at 28 o. C Loading with 2 M DMSO, 10 min at 25 o. C Cooling to -12 o. C at 1 o. C/min Seedling at -12 o. C and holding for 5 min Freezing to -35 o. C at 4 o. C/min Holding at -35 o. C for 5 min Quenching in liquid nitrogen Storage Rapid thawing in 28 o. C water bath Experimental check point

RESULTS n TWO-STEP FREEZING PROTOCOLS l l l n OYSTER EMBRYOS AND EGGS SMALL ABALONE EMBRYOS HARD CLAM AND EGGS VITRIFICATION l l OYSTER EMBRYOS SMALL ABALONE EMBRYOS

MODIFICATION OF THE TWO-STEP PROTOCOL n Direct loading and unloading of 2 M DMSO n Optimizing seeding temperature n Optimizing cooling rate n Shortening holding time n Higher survival rate n Immediate revival of oyster embryos with rotary motion

Survival of 4 -Hr. oyster embryos at consecutive steps during freezing at a freezing rate of 2 o. C/min

Comparisons on the effect of adding trehalose to DMSO

Survival of 5 -Hr. hard clam embryos at consecutive steps during freezing at a freezing rate of 2 o. C/min

Comparisons on the effect of using dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) as CPA.

Effect of freezing rate on the survival of cryopreserved oyster embryos loaded with 2 M DMSO or 2 M glycerol.

Survival of Oyster Embryos at Consecutive Steps of Vitrification Procedure

Survival rates of cryopreserved oyster embryos and eggs

Effect of seeding temperature on the survival of cryopreserved oyster embryos

Schematic of Conventional Two-Step Cryopreservation Procedure Temperature(o. C) Equilibrate in Cryoprotectant 0 Seeding Start slow cooling Quenching in LN 2 Hold for equilibration -196 Time

SUMMARY METHOD SHELLFISH Two-step freezing Vitrification Oyster embryos (Crassostrea gigas) 78 8% 14 8% Hard clam embryos (Meretrix lusoria) 72 10% Unknown Small abalone embryos (Haliotis diversicolor) Negative < 1%

CONCLUSIONS n n n 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 78 8% and 72 10% 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 14 8%, respectively. Both required further optimization of the vitrification procedure.