Eppendorf Piezo-actuated Mouse ICSI (intracytoplasmic sperm injection) - Manual (Page 2)

Animals, media, consumables and devices were used as
described previously [4] with the following modifications:
Media:
CZB-HEPES (CZB-H)
CZB
CZB-HEPES with 12 % polyvinylpyrrolidone (PVP)
Density gradient (e.g. Percoll)
Fluorinert C-77 (FC-77), Fluorinert C-770 (FC-770)
Consumables:
100 μL pipette tips
Piezo impact unit:
Eppendorf PiezoXpert
Userguide No 037 | Page 2
Mouse sperms (fresh or frozen-thawed) are prepared based
on the method of sperm head isolation.
If the sonication method is used, sperms are prepared by
centrifugation and subsequent sonication to isolate sperm
heads by diluting a small sample of sperms with buffer
followed by repeated sonication (e.g. 4 x 15 seconds) [5].
If the piezo assisted method is used, described at 5.2.1,
sperms are prepared using mini swim-up [6] or density
gradient centrifugation.
ICSI dish for sperm head isolation using piezo-assisted
method
2 dishes (A; B) are prepared as shown in Figure 4. In dish
A 2 x 5 μL flat droplets of CZB-HEPES with 12 % PVP are
placed in the center of the dish. One droplet for the sperm
suspension and the other one for the isolated sperm heads
collection. In dish B, a 5 μL droplet of CZB-HEPES with
12 % PVP is placed at the center of the dish and 6 x 5 μL
injection drops of CZB-HEPES are positioned adjacent to
the center. Add approx. five eggs; one in each of the injec-
tion droplets. (One drop is left without cells for cleaning the
capillary.)
First of all sperms are cut in the ‘sperm suspension droplet‘
(dish A). Then the sperm heads are transferred to the
‘sperm heads collection droplet’. Collect as many sperm
heads as possible before transferring them to dish B, where
the injection takes place.
The advantage of this preparation is to minimize the ex-
posure time of oocytes out of the CO
2
incubator and thus
improve the survival rate of oocytes.
Fig. 2: Eppendorf piezo-actuated mouse ICSI system. Worksta-
tion with 2 TransferMan NK 2, PiezoXpert, mounted onto a Ni-
kon Eclipse TE 2000 microscope and incubator GALAXY 14 S.
Fig. 3: ICSI dish with sonicated sperm. Image adapted from [4].
Materials and Equipment
Sperm
droplets
Sperm
suspension
Injection
droplets
head separation methods (sonication method and piezo-
assisted method) are shown as below.
ICSI dish with sonicated sperm
An example with sperm heads that are separated using
sonication is shown in Figure 3 [4]. Here, a 5 μL flat drop of
sperm suspension is positioned in the center of a flat Petri
dish. 3 x 5 μL drops of CZB-HEPES with 12 % PVP are
positioned at one end of the dish along the midline. In addi-
tion, 2 x 5 μL injection drops of CZB-HEPES are placed at
the other end of the dish along the midline. Add approx. five
eggs in one of the injection droplets. Cover with mineral oil.
Methods
1 Preparation of spermatozoa
The arrangement of the drops in the microinjection dish de-
pends on personal preferences. Examples for different sperm
2 Preparation of oocytes
The metaphase II oocytes are collected from superovulated
females and further treated as described previously [4]. The
cumulus-free oocytes are transferred to a culture dish [4].
3 Preparation of the microinjection dish