Semen handling and processing

Question 1

Do sperm kept at room temperature need anything added to them?

Answer 1

Dont need egg yolk or glycerol if sperm is used immediately

Question 2

Cooling sperm

Answer 2

Inhibits bacterial growth, slows down all metabolic processes

Question 3

Motility of sperm that has been rapidly cooled?

Answer 3

Motility low

Question 4

Motility of sperm that has been cooled slowly?

Answer 4

Motility higher than sperm cooled rapidly

Question 5

When should you add egg yolk to sperm?

Answer 5

When cooling sperm as it protects the cell membrane

Question 6

Why should you add glycerol to sperm?

Answer 6

Used to maximise sperm motility after thawing

Lowers freezing point of solution and reduces the amount of intracellular ice formation

Question 7

Rapid cooling of sperm

Answer 7

Sperm just shaking - motility changes - due to membrane damage - fertility affected

Question 8

Hypotonic

Answer 8

Water going into cell

Question 9

Hypertonic

Answer 9

Water leaving cell

Question 10

What is the optimal pH of sperm

Answer 10

Neutral range

Question 11

Isotonic solutions are good for sperm, so when should a slightly hypertonic solution be used?

Answer 11

When freezing sperm so some water leaves cell before freezing. This prevents membrane rupture as water expands upon freezing

Question 12

Why use a buffer in solution with sperm?I

Answer 12

If no buffer is used, metabolic by products build up, becomes acidic and sperm die

Question 13

What kind of sugar does sperm need?

Answer 13

Simple sugars

e.g. glucose, fructose

Sodium chloride - bad - sperm would die, no nutrition

Question 14

Osmolarity

Answer 14

The concentration of a solution, often defined as osmols/L

Question 15

Tonicity

Answer 15

The osmotic pressure of a solution, often expressed as relative to an intercellular environment (e.g. hypo, iso or hypertonic)

Question 16

Osmolarity and thus tonicity changes as water freezes

Answer 16

as water freezes salt can no longer be dissolved – falls out of solution- remaining water becomes more concentrated – bad for cells dehydrated cells

Question 17

Cool very slowly

Answer 17

o ice formation on the outside of the cell starts to occur, salts and sugars fall out of solution, cell becomes very concentrated and tries to adjust by sending water from the interior to exterior of the cell (tries to dilute the environment around it) cell becomes dehydrated and dies

Question 18

Cool very quickly

Answer 18

exterior to the cell freezes very quickly which doesn’t give the cell time to send water out, so all of the water stays inside the cell, but when water freezes it expands which means the cell expands and the cell dies as there is too much physical pressure

Question 19

Optimal cooling rate

Answer 19

o allows for some water to leave the cell (small level of dehydration to occur) which means the cell can expand when ice freezes without dying

Question 20

Small cell (sperm) - cooling rate

Answer 20

= large SA:V ratio - doesn’t contain much water and can react quickly (send water out of it’s cell quickly)

High freezing rate

Question 21

Large cell (embryo) - cooling rate

Answer 21

= Small SA:V ratio - Reacts more slowly, harder for larger cell to remove water

slow cooling rate

Question 22

Cryoprotectants

Answer 22

Increase cryosurvival of cells
Decrease the freezing point of a solution
Maintain membrane fluidity

Influence the response of water to decrease in temperatures

Trade-off between cryoprotection and toxicity (as cryoprotectants are toxic and can kill cells)

Question 23

Penetrating cryoprotectants

Answer 23

Small water soluble molecules
Stay in solution at low temperature
Buffers solute concentration increase – reducing slow cooling injury

E.g. DMSO, glycerol

Question 24

DMSO

Answer 24

Permeates rapidly

Used for embryo freezing

Question 25

Glycerol

Answer 25

Permeates relatively slowly

Used for sperm frezing

Question 26

High concentrations of glycerol

Answer 26

cells do better with slow cooling rates

Question 27

Low concentrations of glycerol

Answer 27

Cells do better with faster cooling rates

Question 28

Non-penetrating cryoprotectants

Answer 28

Large water soluble molecules

Reduce fast cooling injury

e.g. sucrose , egg yolk

Question 29

Sucrose

Answer 29

Enhances effect of penetrating cryoprotectant

Question 30

Egg yolk

Answer 30

Improves membrane fluidity

low density lipoproteins associate with the sperm membrane to stabilise the structure and prevent loss of lipids from the membrane during freezing

Question 31

Vitrification

Answer 31

Freezing in the absence of ice

Solidification of liquid without crystallisation (instead, with formation of glass)

Achieved by:
Replacement of water within cell with cryoprotectant (usually has to be a penetrating cryoprotectant e.g. DMSO)
Ultra rapid cooling rates – don’t have to worry about the formation of ice crystals within cell

Much more successful then slow freezing

Question 32

Why/Why not use vitrification?

Answer 32

Pros:
Improved survival rates
Application to whole tissue not just cells

Cons:
Technically difficult
Questionable biosecurity

Question 33

Can diluents be stored?

Answer 33

They be stored briefly in the refrigerator but are usually used fresh

Question 34

For fresh use of sperm how many mls are used?

Answer 34

0.2ml

Question 35

Mixing of diluent and sperm

Answer 35

Diluent is ALWAYS added to sperm not the other way around

Question 36

How long can diluted semen be stored in a refrigerator?

Answer 36

Up to 24 hours after this fertility declined

stored at 4 degrees

Question 37

Diluents to be added when freezing sperm?

Answer 37

Cryoprotectant such as glycerol
Egg yolk
pH buffer