Biology Reference
In-Depth Information
Application of new sperm cryopreservation and recovery
Methods
The methods reported here yield fertilization rates of 70±5% with C57BL/6J oo-
cytes and cryopreserved C57BL/6J sperm. This is a significant improvement over
many previously published approaches [2]-[5], but these data, and those reported
by others, may not reflect performance “in the field”. Indeed, the demand for im-
proved sperm cryopreservation methods comes from the necessity to cryopreserve
GM lines, not inbred strains. Thus, before reaching conclusions on applicability,
we believe it is crucial to test methods as they will be used in practice.
Over a 15-month period (7/19/06 to 10/8/07), sperm were cryopreserved
from 994 GM lines (predominantly knockouts and transgenics) submitted to The
Jackson Laboratory. For the purpose of this study, the predominant genetic back-
ground of the GM line was defined by the inbred strain chosen to be the source of
oocytes for IVF. This approach generally holds true, but more importantly, reflects
the situation encountered by repositories and core facilities responsible for cryo-
preservation. To allow for robust statistical comparisons, only those backgrounds
represented by five or more GM lines were considered, limiting the data set to 735
GM lines and 12 predominant genetic backgrounds.
Sperm were cryopreserved as described previously and stored in liquid nitro-
gen for at least 24 hours. In vitro fertilization was performed using 60 minutes
of sperm pre-incubation and oocytes from superovulated inbred females corre-
sponding to the predominant genetic background of each of the various GM
lines. For comparison, IVF data was obtained using freshly collected sperm from
847 GM lines being maintained on the same genetic backgrounds. No difference
in fertility was detected between freshly collected or cryopreserved sperm for 10
of the 12 genetic backgrounds (129S1/SvImJ; C57BLKS/J; 129X1/SvJ; BALB/cJ;
B6129SF1/J; NOD/ShiLtJ; FVB/NJ; C3H/HeJ; C3HeB/FeJ; DBA/2J; Figure
3a). Cryopreserved sperm from two of the genetic backgrounds (C57BL/6J and
BALB/cByJ) showed slight reductions in fertility. However, when the proportion
of 2-cell embryos generated on these two backgrounds using our new method
and a method modified from Sztein et al[4] were compared, six- and five-fold
increases were observed, respectively (Figure 3a vs Table 1). When frequency dis-
tributions are compared, a similar proportion of lines perform poorly (defined as
fertilization rate of five percent or less) whether fresh or frozen sperm are used,
demonstrating the reliability of the method. The difference observed between the
fertilization capacity of the GM C57BL/6 lines (Figure 3a, Table 1) and inbred
C57BL/6J mice (Figure 1a, Figure 2) is likely due to genetic modifications that
may have affected fertility and the presence of alleles from other strains in lines
that were not fully congenic. Relying on data from inbred strains alone would
have overestimated the capability of the method in practice.
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