Conserving, Distributing and Managing Genetically Modified Mouse Lines by Sperm Cryopreservation - Genetic Engineering: Recent Developments in Applications - page 64

here, sperm were consistently cooled at the same rate. The warming rate also af-
fects sperm survival[11], and encouraging results have been reported when thaw-
ing at 54°C[12]. We compared thawing cryopreserved C57BL/6J sperm at 37°C
and 54°C. No differences in sample warming rate or sperm survival were observed
(Table 2). However, a greater proportion of oocytes developed into 2-cell embryos
when fertilized in vitro with sperm thawed at 37°C (Table 2). Even though the
straws were exposed to the 54°C water bath for only 5 sec, the outer layers of the
straw may have warmed to 54°C before the contents in the center had time to
equilibrate. Those cells exposed to 54°C and to temperatures above 37°C likely in-
curred varying degrees of thermal damage, reducing their ability to fertilize. This
mechanism is supported by the assertion of Jiang et al[13] that thawing sperm at
37°C reduces the risk of thermal damage.
Table 2. Suitable cooling and warming rate determination.
Promoting/Maintaining Fertilization capacity
Improved IVF success has been reported with cryopreserved C57BL/6 sperm[12],
[14]. One characteristic shared by these reports is a processing step that delayed
the time between thawing and combining eggs and sperm. To determine if pre-
incubating cryopreserved C57BL/6J sperm enhances fertilizing ability, fresh and
cryopreserved sperm samples were incubated for 0, 30, 60, or 80 min in Mouse
Vitro Fert medium (MVF; Cook Medical; Brisbane, Australia). Freshly collected
C57BL/6J sperm did not benefit from incubation. However, cryopreserved sperm
exhibited considerable enhancement, with maximum fertility of cryopreserved
C57BL/6J sperm being achieved after one hour of incubation (Figure 1a). Thus,
after thawing, cryopreserved C57BL/6J sperm acquire fertilization competence