Semen handling and processing Flashcards
Do sperm kept at room temperature need anything added to them?
Dont need egg yolk or glycerol if sperm is used immediately
Cooling sperm
Inhibits bacterial growth, slows down all metabolic processes
Motility of sperm that has been rapidly cooled?
Motility low
Motility of sperm that has been cooled slowly?
Motility higher than sperm cooled rapidly
When should you add egg yolk to sperm?
When cooling sperm as it protects the cell membrane
Why should you add glycerol to sperm?
Used to maximise sperm motility after thawing
Lowers freezing point of solution and reduces the amount of intracellular ice formation
Rapid cooling of sperm
Sperm just shaking - motility changes - due to membrane damage - fertility affected
Hypotonic
Water going into cell
Hypertonic
Water leaving cell
What is the optimal pH of sperm
Neutral range
Isotonic solutions are good for sperm, so when should a slightly hypertonic solution be used?
When freezing sperm so some water leaves cell before freezing. This prevents membrane rupture as water expands upon freezing
Why use a buffer in solution with sperm?I
If no buffer is used, metabolic by products build up, becomes acidic and sperm die
What kind of sugar does sperm need?
Simple sugars
e.g. glucose, fructose
Sodium chloride - bad - sperm would die, no nutrition
Osmolarity
The concentration of a solution, often defined as osmols/L
Tonicity
The osmotic pressure of a solution, often expressed as relative to an intercellular environment (e.g. hypo, iso or hypertonic)
Osmolarity and thus tonicity changes as water freezes
as water freezes salt can no longer be dissolved – falls out of solution- remaining water becomes more concentrated – bad for cells dehydrated cells
Cool very slowly
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
Cool very quickly
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
Optimal cooling rate
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
Small cell (sperm) - cooling rate
= large SA:V ratio - doesn’t contain much water and can react quickly (send water out of it’s cell quickly)
High freezing rate
Large cell (embryo) - cooling rate
= Small SA:V ratio - Reacts more slowly, harder for larger cell to remove water
slow cooling rate
Cryoprotectants
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)
Penetrating cryoprotectants
Small water soluble molecules
Stay in solution at low temperature
Buffers solute concentration increase – reducing slow cooling injury
E.g. DMSO, glycerol
DMSO
Permeates rapidly
Used for embryo freezing
Glycerol
Permeates relatively slowly
Used for sperm frezing
High concentrations of glycerol
cells do better with slow cooling rates
Low concentrations of glycerol
Cells do better with faster cooling rates
Non-penetrating cryoprotectants
Large water soluble molecules
Reduce fast cooling injury
e.g. sucrose , egg yolk
Sucrose
Enhances effect of penetrating cryoprotectant
Egg yolk
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
Vitrification
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
Why/Why not use vitrification?
Pros:
Improved survival rates
Application to whole tissue not just cells
Cons:
Technically difficult
Questionable biosecurity
Can diluents be stored?
They be stored briefly in the refrigerator but are usually used fresh
For fresh use of sperm how many mls are used?
0.2ml
Mixing of diluent and sperm
Diluent is ALWAYS added to sperm not the other way around
How long can diluted semen be stored in a refrigerator?
Up to 24 hours after this fertility declined
stored at 4 degrees
Diluents to be added when freezing sperm?
Cryoprotectant such as glycerol
Egg yolk
pH buffer
