Lab Practical Exam Flashcards
Describe how you carry out a wet preparation for semen analysis?
- Keep sperm incubated
- Examine colour, pH, viscosity and volume.
- Take 10uml and examine under a phase contrast microscope
- Count the numbers of motile sperm (200 in 4 fields of vision)
- Examine for the presence of other cells and debris
What counts as a type A (Rapidly progressively motile) sperm?
Velocity of more than 25 um/s
What counts as a type B (slow progressive cell)
Velocity of 5 - 24 um/s
What speed in a class c moving at?
Velocity of less than 5 um/s
What is the normal pH of semen?
7.2 - 7.8
How many days abstinence do you need to get a good quality sample?
Between 3 and 7
If the semen analysis seems abnormal how long should you wait before taking a new one and why?
3 months. This is how long the entire process of spermatogenesis takes. A cold/bing drink 3 months before the sample can affect it.
What is the density of a mature, morphologically normal sperm?
1.12 g/ml
What is the density of an abnormal sperm?
1.06 - 1.09 g/ml
What is the density of the 40% solution used in density graded centrifugation?
1.06g/ml
What is the density of the 80% solution used in density graded centrifugation?
1.10g/ml
Describe how you carry out a density graded centrifugation sperm preparation
- Add 1ml 80% solution to the bottom of the test tube
- Add 1ml 40% solution
- Add semen on top slowly
- Centrifuge at 300 xg for 20 mins
- Aspirate the upper raft (contains the seminal plasma)
- remove the 40% layer and the lower interface raft whilst leaving most of the lower 80% layer in place.
- Transfer the pellet (75ul) into a single clean 15ml centrifuge tube and wash with 4ml sperm wash.
- Centrifuge at 300xg for 5 minutes
- Aspirate most of the upper supernatant and leave around 1ml to examine and use.
Why are normal sperm more dense?
Their DNA is more condensed
Also abnormal cells eg those with round heads are much less dense so don’t make it down to the bottom.
List some drawbacks of density graded centrifugation
- Overcentrifugation can cause a build up of reactive oxygen species.
- Overloading the gradient can block the interface layer and prevent the passage of functional sperm.
- Large amount of debris also collected (not the same in swim up where you get a small number of highly motile cells)
What are some of the advantages of density graded centrifugation?
- Suitable for all samples including very viscous samples
- Get a higher number of cells.
- Faster than swim up
What temperature are sperm cryopreserved at?
- 196 degrees
What are the three main risk to cells during cryopreservation?
Mechanical damage from ice crystal formation
Osmotic stress
Cold shock injury
Why do sperm freeze better than eggs?
Eggs are larger and are more prone to ice crystal formation
Why do sperm cryopreserve relatively well?
- They are small
- Large surface area to volume ratio
- Plasma membrane is highly permeable to water which facilitates dehydration when a cryoprotectant is added.
- The sperm head is mostly condensed DNA and contains little cytoplasm making them less susceptible to ice crystal formation.
What is the cryoprotectant added to sperm and how does it work?
Glycerol (egg yolk or soy have also been used)
Cell permeable so enters the cell and lowers the freezing point of intracellular water to induce greater dehydration at lower temperatures and reduce ice crystal formation.
Roughly what % of motile sperm are lost during cryopreservation?
60 - 90%
Why, in the clinic re samples stored in vapour rather than liquid nitrogen?
To reduce the risk of infection spread
How is sperm prepared following its removal from liquid nitrogen?
Thawed rapidly in a room temperature water bath
Slow dilution with sperm wash
Density graded centrifugation
Describe what is done before the catheter is passed through in an embryo transfer procedure
- Ensure the bladder is appropriately full
- Optimise the transabdominal scan of the uterine cavity
- Remove cervical mucus with cotton buds
