L1 pt. 2: Cryopreservation

Question 1

Give the 3 steps of cryopreservation:

Answer 1

• Cooling (gradual to avoid cold shock; often includes equilibriation step)
• Freezing (cryopreservants such as glycerol added in advance)
• Thawing

Question 2

How do cells respond to inclusion of cryoprotectants?

Answer 2

• Solution osmolarity rapidly raised -> initially, cells shrink
• Later, original volume is restored following re-equilibriation

Question 3

2 key effects of sperm freezing on sperm quality:

Answer 3

• Reduces number of surviving spermatozoa
• Reduces competency of survivors (e.g. in frozen-thawed bull sperm, a 5 fold increase in sperm number is required to achieve high fertility compared with fresh samples, with accompanying decrease in duration of viability)

Question 4

What factors govern optimal cooling rate?

Answer 4

• High solution effects at lower cooling rate
• High intracellular ice levels produced with faster cooling rate
• Intermediate rate balances these two effects

Question 5

List the 4 requirements for successful sperm cryopreservation:

Answer 5

• Majority of spermatozoa should retain an intact plasma membrane (typically ~50% do not)
• Cell function in the ‘live’ population should not be impaired (usually causes shortened lifespan)
• All organelles should be intact and functional
• Sperm DNA should be intact and able to support development

Question 6

How can cold shock damage sperm?

Answer 6

• Cold shock/slow cooling causes a form of premature capacitation
• Reduced survival time

Question 7

Give examples of additives used in sperm cryopreservation:

Answer 7

• Egg yolk (routinely used) -> Issue of disease risk due to animal origin
• Skimmed milk
• Soybean lecithin
• Coconut milk
• Various antioxidants

Question 8

How may vitrification improve cryopreservation protocol?

Answer 8

• Vitrification: Glass formation
• If solutions are cooled rapidly enough, they do not form ice crystals
• Has typically involved such high cryopreservant concentrations that sperm cannot survive initial dilution
• Investigation into protocol of direct vitrification in liquid nitrogen (without cryopreservants) have shown good levels of post-thaw motility and viability

Question 9

Sources of contamination in vitrification cryopreservation protocol:

Answer 9

• Liquid nitrogen (not sterile) -> e.g. contaminated tanks
• Semen (not sterile)
• Microorganisms thus preserved with sample when present

Question 10

Why is glycerol useful as a cryopreservant:

Answer 10

• Interferes with ice crystal formation

Question 11

What alteration to cryopreservation can be beneficial in particularly poor samples?

Answer 11

• Carry out multi-step dilution when adding cryopreservants