Biology Reference
In-Depth Information
Widespread adoption of sperm cryopreservation will hopefully make the pro-
active cryopreservation of strains routine. This will benefit facilities and investiga-
tors by reducing operating costs, providing protection against disease outbreaks
or disasters, and improving collaboration by reducing barriers to distribution. A
system in which investigators cryopreserve lines and deposit the cryopreserved
material within one of the many publicly funded resource centers may now be
possible. This will reduce the operating costs of resource centers, provide secure
off-site backup of lines, and relieve investigators and core facilities of the burden
of maintaining and distributing lines. The development of an effective, efficient
sperm cryopreservation method marks a new paradigm in repository operations,
facilitating the global need to archive and distribute mouse models.
Methods
Animals
Mice were maintained at The Jackson Laboratory (Bar Harbor, ME, USA) in
accordance with The Jackson Laboratory institutional protocols and the Guide
for the Care and Use of Laboratory Animals [26]. The animals were housed in
a minimum barrier facility with a light cycle of 14 hrs on and 10 hrs of (on at
05:00 AM).
sperm collection and cryopreservation
Briefly, 2 mL of CryoProtective Medium [CPM - 18% w/v raffinose (Sigma Al-
drich; cat # R7630); 3% w/v skim milk (BD Diagnostics; cat # 232100); 477 µM
monothioglycerol (Sigma cat # M6145); water (Invitrogen, cat # 15230-238)]
were used to collect the sperm from the epididymides and vas deferentia of two
3-5 month-old male mice. Sperm were allowed to disperse from the tissue for
10 min and then 10 µL of sperm+CPM and ballast were loaded into 0.25 mL
French straws (IMV; Maple Grove, MN; cat# AAA201). Straws were sealed with
an impulse heat sealer (American International Electric; Whittier, CA; model
AIE-305HD) and five straws per cassette (Zanders Medical Supplies; Vero Beach,
FL; cat #16980/0601) were placed onto a raft (polystyrene 15.5 cm wide×20 cm
long×2.5 cm deep) floating in LN2 for ≥10 min. This resulted in the sperm being
cooled from −10 to −60°C at a rate of 37±1°C/min before being plunged and then
stored in LN2.
Cooling rates were determined by placing the wire lead of an Ertco TC4000
thermocouple (Dubuque, IA) into the 10 µL volume of sperm+CPM within a
straw that was inside a cassette with 5 loaded straws. Data points were obtained
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