Basic information on the cryopreservation process kos Horvth - - PowerPoint PPT Presentation

DEPARTMENT OF AQUACULTURE

Basic information on the cryopreservation process

Ákos Horváth Department of Aquaculture, Szent István University, Gödöllő, Hungary

COST Action FA1205 AQUAGAMETE 5th AQUAGAMETE Training School Valencia, Spain, 7-11th March, 2016

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

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

• Water supercools below the

freezing point

• Ice formation starts along ice

nuclei

• Primarily water molecules are

incorporated into the ice crystals

Temperature (°C)

• 160
• 120
• 80
• 40

40

Time (seconds)

20 40 60 80 100 120 140 160 180 200 220 240

Distilled water Extender + cryoprotectant

DEPARTMENT OF AQUACULTURE

Cryopreservation: cooling of live cells, vitrification, the role of cryoprotectants

H2O H2O H2O Slow cooling Thawing Fast cooling Thawing Thawing Optimum cooling H2O H2O Thawing Ultra-fast cooling

Case 1. Case 2. Case 3. Case 4.

DEPARTMENT OF AQUACULTURE

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

• Others: dimethyl sulfoxide

(DMSO), dimethyl acetamide (DMA)

–Roles

• Membrane

stabilization

• Inhibition of ice

crystallization

• Lowering of the

freezing point

DEPARTMENT OF AQUACULTURE

Cryopreservation of fish sperm – the role of extenders, cryoprotectants and dilution ratios

• 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

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

• Cooling in the vapor
• f liquid nitrogen

–Styrofoam box

• Simplicity
• Low cost

–Computer-controlled

freezer

• Controlled conditions
• More reliable replication

Cryopreservation of fish sperm – methods, straws, cooling rates

DEPARTMENT OF AQUACULTURE

Cryopreservation of fish sperm – methods, straws, cooling rates

Temperature (°C)

• 180
• 135
• 90
• 45

45

Time (sec)

60 120 180 240 300

5 ml straws 1.2 ml staws 0.5 ml straws

DEPARTMENT OF AQUACULTURE

Cryopreservation of fish sperm – storage, thawing

• Storage

– Dewars:

• Large capacity
• Extended

storage time

• Shipping dewars

– Other storage

devices

• Goblets
• Canes
• Thawing

– Typically at 40°C

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

Cryopreservation of fish sperm – fertilization with cryopreserved sperm

Hatch (%)

10 20 30 40

Storage time before fertilization

2 days 1 day 2 hours no storage control 32 21 31 37 6

DEPARTMENT OF AQUACULTURE

Prediction of sperm quality without fertilization

Post-thaw motility (%)

10 20 30 40 50 60 70 80 90 100

5% MeOH 10% MeOH 15% MeOH 5% DMSO 10% DMSO 15% DMSO control

Fertilization (%)

25 50 75 100 5% MeOH 10% MeOH 15% MeOH 5% DMSO 10% DMSO 15% DMSO control

Motility is not always a good predictor of fertilizing capacity

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

Genetic damage associated with cryopreservation

DEPARTMENT OF AQUACULTURE

Cryopreservation of fish eggs and embryos

• Problems:

–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

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

• Cryopreservation of

larvae is possible

• Best larval stages

for cryopreservation are trochophores and veligers

• Very slow cooling

rates

Cryopreservation in other aquatic species

DEPARTMENT OF AQUACULTURE

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

DEPARTMENT OF AQUACULTURE

Further development

• Cryopreservation and transplantation of primordial germ cells (PGCs)
• Cryopreservation and transplantation of undifferentiated type A spermatogonia

Okutsu et al. 2007 Science, 317, 1517.