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Approximately 75% of C57BL/6J sperm do not survive cryopreservation
(Table 2). Consequently, it may be necessary to increase sperm concentration
to ensure that enough viable sperm are present to maximize fertility. How-
ever, this could also increase the concentration of dead sperm, which may be
detrimental[19]. This hypothesis is supported by reports demonstrating improved
fertilization following the separation of viable from non-viable sperm prior to
IVF[12], [15]. To determine whether the requirement for a pre-incubation was
linked to limited concentrations of viable sperm or increased concentrations of
dead sperm, freshly collected sperm were a) diluted to limit the number of live
sperm or b) were mixed with dead sperm to simulate the proportion and concen-
tration of dead sperm present following cryopreservation and thawing. Sperm in
the various treatments were incubated for 0 or 60 min before C57BL/6J oocytes
were added. The proportion of oocytes developing into 2-cell embryos was deter-
mined after overnight incubation.
The percentage of oocytes developing into 2-cell embryos did not differ be-
tween 0 and 60 min for Control (Figure 1b), confirming our previous observation
that freshly collected C57BL/6J sperm do not require incubation prior to being
combined with oocytes. Further, no difference in the proportion of oocytes devel-
oping into 2-cell embryos was detected between Control and Diluted live sperm
at time 0 or 60 min (Figure 1b). Thus, a broad range of viable sperm concentra-
tions yield similar IVF results and reduced sperm concentrations do not lead to
a requirement for sperm pre-incubation. Lastly, we determined if dead sperm
or substances released from sperm damaged by cryopreservation interfere with
fertilization. The percentage of oocytes developing into 2-cell embryos using Di-
luted live sperm was compared with Diluted live+dead sperm. No difference was
detected between the two treatments at time 0 (Figure 1b), indicating that dead
sperm and the substances they release are not responsible for the initial reduction
in fertilization capacity of cryopreserved C57BL/6J sperm. In contrast, after 60
min of sperm incubation, the Diluted live+dead sperm fertilized ~26% fewer oo-
cytes than the Diluted live sperm (Figure 1b). This demonstrates that dead sperm
or substances they liberate can be deleterious to the fertilizing capacity of viable
sperm following prolonged exposure. Based on these results, it can be concluded
that the benefit of a pre-incubation step following thawing is separable from the
effects of limiting viable sperm concentrations or increasing the concentration of
dead sperm.
While the generation of ROS is necessary for fertilization[20], overproduction
of ROS during cryopreservation[21] could alter this process or damage sperm,
impairing their ability to fertilize or to support subsequent embryo development.
To determine if reducing ROS generation can increase the fertilizing capacity of
C57BL/6J sperm, cryopreservation was carried out in the presence of varying
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