Basic information on the cryopreservation process

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1 COST Action FA1205 AQUAGAMETE 5 th AQUAGAMETE Training School Valencia, Spain, 7-11 th March, 2016 Basic information on the cryopreservation process Ákos Horváth Department of Aquaculture, Szent István University, Gödöllő, Hungary

2 Summary Cryopreservation: basics of cryopreservation cooling of water and aqueous solutions Cryopreservation: cooling of live cells, vitrification, the role of cryoprotectants Cryopreservation of fish sperm the role of extenders, cryoprotectants and dilution ratios Cryopreservation of fish sperm methods, straws, cooling rates Cryopreservation of fish sperm storage, thawing Cryopreservation of fish sperm fertilization with cryopreserved sperm Commercial application reasons of failure

3 Cryopreservation: basics of cryopreservation cooling of water and aqueous solutions

4 Cryopreservation: basics of cryopreservation cooling of water and aqueous solutions 40 Water supercools below the freezing point Ice formation starts along ice nuclei Temperature ( C) Primarily water molecules are incorporated into the ice crystals Time (seconds) Distilled water Extender + cryoprotectant

5 Cryopreservation: cooling of live cells, vitrification, the role of cryoprotectants H 2 O Case 1. Slow cooling Thawing H 2 O H 2 O Case 2. Fast cooling Thawing H 2 O Case 3. Optimum cooling Thawing H 2 O Case 4. Ultra-fast cooling Thawing

6 Cryopreservation: cooling of live cells, vitrification, the role of cryoprotectants Cryoprotectants External cryoprotectants Sugars (glucose, fructose, sucrose, trehalose) Polymers (polyvinyl pyrrolidone) Internal cryoprotectants Alcohols methanol Polyols ethylene glycol, propylene glycol, glycerol Roles Membrane stabilization Inhibition of ice crystallization Lowering of the freezing point Others: dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA)

7 Cryopreservation of fish sperm the role of extenders, cryoprotectants and dilution Estimation of motility Visual CASA Extenders solution of sugars and salts Cryoprotectants most often DMSO or methanol Dilution ratios: from 1:1 to 1:9 ratios

8 Cryopreservation of fish sperm methods, straws, cooling rates Cooling in pellets: In a block of dry ice Requires a thawing medium Cooling in straws: Most common technique Used in most livestock species Cryovials Glass capillaries

9 Cryopreservation of fish sperm methods, straws, cooling rates Cooling in the vapor of liquid nitrogen Styrofoam box Simplicity Low cost Computer-controlled freezer Controlled conditions More reliable replication

10 Cryopreservation of fish sperm methods, straws, cooling rates 45 0 Temperature ( C) ml straws 1.2 ml staws 0.5 ml straws Time (sec)

11 Storage Cryopreservation of fish sperm storage, thawing Dewars: Large capacity Extended storage time Shipping dewars Other storage devices Goblets Canes Thawing Typically at 40 C

12 Cryopreservation of fish sperm fertilization with cryopreserved sperm Determination of post-thaw motility varies with species General rules of fertilization are similar to those with fresh sperm Effective sperm:egg ratios start from 5000 spermatozoa to 1 egg Research for the prediction of sperm quality without fertilization

13 Cryopreservation of fish sperm fertilization with cryopreserved sperm Hatch (%) days 1 day 2 hours no storage control Storage time before fertilization

14 Prediction of sperm quality without fertilization Post-thaw motility (%) % MeOH 10% MeOH 15% MeOH 5% DMSO 10% DMSO 15% DMSO control Fertilization (%) Motility is not always a good predictor of fertilizing capacity 0 5% MeOH 10% MeOH 15% MeOH 5% DMSO 10% DMSO 15% DMSO control

15 Prediction of sperm quality without fertilization Viability: live-dead fluoresecent dual staining combined with flow cytometry Comet-assay: single-cell gel electrophoresis assay Computer-assisted sperm analysis - CASA

16 Genetic damage associated with cryopreservation

17 Cryopreservation of fish Problems: eggs and embryos Egg envelope Egg activation upon release into a liquid Structure of fish embryos Which embryonic stage to use Limited success Vitrification of flounder embryos Vitrification of carp embryos

18 Cryopreservation in other aquatic species Cryopreservation of bivalvian sperm Differences from fish sperm: Reduced post-thaw motility Reduced fertilization Extender osmolality around 1000 mosmol/ kg

19 Cryopreservation in other aquatic species Cryopreservation of larvae is possible Best larval stages for cryopreservation are trochophores and veligers Very slow cooling rates

20 Commercial application reasons of failure Very few cases of commercial application (if any) Mostly sperm banks maintained by laboratories and research institutions Reasons: Science ahead of industry Adaptation of protocols originally developed for livestock are not suitable for aquaculture Low level of standardization and international cooperation

21 Further development Okutsu et al Science, 317, Cryopreservation and transplantation of primordial germ cells (PGCs) Cryopreservation and transplantation of undifferentiated type A spermatogonia

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