Lab Practical: Sperm Preparation

Question 1

Give the two main methods of sperm preparation

Answer 1

direct swim up from semen

density gradient centrifugation

Question 2

Why is semen preparation required

Answer 2

1. prolonged exposure to seminal plasma adversely affects sperm function e.g. reduces their ability to fertilise an egg
2. to mimic the natural selection characteristics seen in vivo

Question 3

What is the principles of density gradient centrifugation

Answer 3

This method separates the constituents based upon their density or specific gravity.
Cells will distribute trought the gradient according to location in the gradient that matches their density.
Morphologically normal and abnormal spermatozoa have different densities.

Question 4

What are the densities of morphologically normal sperm in general

Answer 4

usually have a density greater than 1.12g/ml

Question 5

What densities do morphologically more abnormal sperm tend to have

Answer 5

between 1.06 and 1.09g/ml

Question 6

What is the most commonly used method of density gradient sperm prep

Answer 6

two layer density gradient, formed by a top layer (40 percent v/v )and a lower layer (80 percent v/v)

Question 7

What solution is used in density gradient centrifugation to separate out sperm

Answer 7

colloid silica preparations ( one is a 40 percent and the other is 80 percent)

Question 8

What is the specific gravity of 40 percent colloid silica solution

Answer 8

1.06g/ml

Question 9

what is the specific gravity of 80 percent colloid silica

Answer 9

1.10gml

Question 10

What do the specifc gravities of the solutions mean in terms of sperm prep

Answer 10

As the sg of the 80 percent solution is 1.10g/ml only sperm which have a comparable specific gravity will penetrate this layer to form the ‘pellet’ of sperm at the bottom of the test tube - therefore only the most mature, normal spermatozoa should penetrate this layer to form the pellet

Question 11

Outline the technique of DGC

Answer 11

Place 1ml of 80 percent gradient at the bottom of a 15ml centrifuge tube
Carefully overlay 1ml of 40 percent
Overlay liquefied semen on top of the 40 percent layer
DO not disturb the layers
Centrifuge at 300g for 30 minutes in a swing out rotor
Aspirate the seminal plasma ‘upper raft’
Remove the 40 percent layer and lower inferface ‘raft’ ; leave most of 80 percent layer in place. discard aspirated material
Transfer the pellet (75uL) into a single clean 15mL centrifuge tube ad wash with 4mL sperm wash
Centrifuge at 300g for 5 mins
Carefully aspirate most of the supernatant leaving around 1ml of supernatant.

Question 12

Describe the principles of DSUS

Answer 12

Liquified semen is layered beneath a culture medium and during subsequent incubation period the progressively motile sperm migrate from the semen layer into the culture medium

Question 13

Why is DSUS used

Answer 13

Given that progressive motility is required for sperm to swim up into the culture medium, it is though that this method mimics the process by which sperm escape the liquefied ejaculate ad colonize the cervical mucus.
DSUS is thought to be more comparable to the in vivo process than other sperm prep techniques.

Question 14

What factor is important for progressive motility

Answer 14

temperature - affects PM greatly

Question 15

Outline the procedure of DSUS

Answer 15

Label round bottom 14ml culture tube with name
Place 0.6Ml of prewarmed sperm wash in bottom of the tube
Using a sterile pipette, carefully underlay 300uL of well mixed, liquefied semen into the bottom of the tube
Cape tube and place at 45 degree angle in a tube rack in the 37 degree incubator for 30-60 minutes
Using a sterile pipette remove 2/3 of the culture medium layer (around 400uL)
Do not disturb the inferface. Discard if inferface is disturbed
Transfer the aspirated upper layer into a 15mL centrifuge tube containing 4mL sperm wash buffer
Centrifuge for 0.3 RCF for 5 minutes
Aspirate the supernatant leaving 0.5 mL and resuspend the pellet.

Question 16

What two important aspects of DSUS preparation is it important to adhere to in order to improve the technique

Answer 16

Incubate the tube etc to maintain consistant temperature of 37 degrees- temp affects motility greatly
Angle tube at 45 degrees- gives greater surface area for swimming

Question 17

Give the differences between DSUS and density gradient

Answer 17

DSUS:
takes longer
more selective of progressive motility
less debris
less concentrated

DGC:
may damage sperm
selects based on density and morphology rather than motility
there is rafting and debris
more concentrated sample

Question 18

In which circumstances is each technique typically used (according to WHO guidelines)

Answer 18

DSUS- normozoospermic samples
DGC- oligozoospermic

(in reality in Ninewells all samples are prepared using DGC)