Eppendorf Piezo-actuated Mouse ICSI (intracytoplasmic sperm injection) - Manual (Page 5)

Userguide No 037 | Page 5
programs can be stored. Each program consists of param-
eter set A and B. Usually, parameter set A is used for the
penetration of the zona pellucida and set B is used
for the penetration of the oolemma. Both sets A and B can
be triggered via either the button on the control unit or the
foot control.
Optimization of parameters:
1. Set the parameters for the speed and number of pulses to 1.
2. Gradually increase the value for intensity (starting from 1)
until the piezo impulses are strong enough to penetrate the
membranes.
3. Fine tune the speed and pulse parameters.
4. Use the lowest parameter settings that work.
Usually, the parameter settings for Fluorinert are slightly higher
than for mercury.
Position the sperm so that its neck is at the opening of the
capillary (Figure 9A).
Then apply a few pulses to separate the head from the tail.
Aspirate the head into the injection capillary (Figure 9B). Repeat
the procedure with the next sperm. Separate as many sperm
heads as possible
Transfer the separated sperm heads into the ICSI dish before
injection (see Figure 4B).
5.2.2 Microinjection of sperm heads into oocytes:
Aspirate up to five sperm heads into the capillary. This way is
more efficient and less time consuming than treating one sperm
at a time. Make sure the sperm heads are not directly lined up
adjacent to each other within the capillary to avoid accidentally
injecting more than one sperm head at a time into an oocyte.
A
Parameter set A
(zona penetration) Parameter set B
(oolemma penetration)
Intensity 10 1
Speed 1 1
Pulse ∞ 1
B
Parameter set A
(zona penetration) Parameter set B
(oolemma penetration)
Intensity 20 5
Speed 1 1
Pulse ∞ 1
Table 1: Parameter settings for mouse ICSI using mercury (A) and Fluorinert (B).
NOTE: Users should optimize the parameters for their own
experiments as the settings always depend on individual labo-
ratory protocols. As a guideline, if no cryopreserved oocytes
are used, we recommend the parameters for both mercury and
Fluorinert respectively, shown in Table 1.
For sperm head separation using the PiezoXpert, parameters
with higher intensity and speed can be used. These parameters
can be saved e.g. in program 2.
5.2 During ICSI
5.2.1 Isolation of sperm head:
When using the sonication method, refer to 1.
For applying the piezo-assisted method: First aspirate a single,
motile sperm (head first) into the injection capillary (using the
ICSI dish for sperm head isolation (Figure 4A).
Fig. 9: Immobilization of a sperm using PiezoXpert (A, B)
A
B