1. 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.

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

3. OSMOMETRIC CHARACTERISTRICS  EQUILIBRIUM OSMOMETRIC BEHAVIOR  NON-EQUILIBRIUM OSMOMETRIC BEHAVIOR HYDRAULIC CONDUCTIVITY CRYOPROTECTANT PERMEATION  MODELING AND PREDICTION

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

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

6. Comparison of cell volume fluctuation in small abalone eggs during propylene glycol loading and unloading with or without osmotic buffer.

7. Comparison of intracellular CPA concentration in small abalone eggs during propylene glycol loading and unloading with or without osmotic buffer.

8. TOXICITY TOLERANCE TO CPA  TIME  TEMPERATURE  CONCENTRATION  TYPES OF CPA  DEVELOPMENT STAGE

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

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

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

12. Toxicity tolerance of oyster embryos of various development stages to propylene glycol (PG) of various concentrations

13. INTRACELLULAR ICE FORMATION CHARACTERISTRICS  TEMPERATURE DEPENDENCY  TIME DEPENDENCY  EFFECT OF CPA  MODELING AND PREDICTION

14. CRYOPRESERVATION PROTOCOL

15. Two-step Freezing Procedure of Oyster and Hard Clam Embryos 4-Hr. oyster embryos cultured at 28 o C Loading with 2M 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

16. Two-step Freezing Procedure of Oyster and Hard Clam Embryos 4-Hr. oyster embryos cultured at 28 o C Loading with 2M 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

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

18. MODIFICATION OF THE TWO-STEP PROTOCOL n Direct loading and unloading of 2M 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

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

20. Comparisons on the effect of adding trehalose to DMSO

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

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

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

24. Survival of Oyster Embryos at Consecutive Steps of Vitrification Procedure

25. Survival rates of cryopreserved oyster embryos and eggs

26. Effect of seeding temperature on the survival of cryopreserved oyster embryos

27. Schematic of Conventional Two-Step Cryopreservation Procedure Temperature( o C) Time 0 Equilibrate in Cryoprotectant Seeding Start slow cooling Hold for equilibration Quenching in LN2 -196

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

29. CONCLUSIONS 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.