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over time. Similar findings have been reported with sperm incubated in media
containing methyl-beta-cyclodextrin[15]. Together, these studies indicate that
cryopreserved C57BL/6 sperm must undergo maturational events prior to the ad-
dition of oocytes. It is postulated that sperm must commence maturation prior to
the addition of oocytes because of the spontaneous cortical granule release, which
results in zona hardening and fertilization capacity reduction[16], [17]. In fact,
we have observed strain specific differences in the time required for zona pellucida
dissolution (personal unpublished data), which may be due to differences in the
level of spontaneous cortical granule release or may simply reflect differences in
the zona pellucidae from different strains. Nonetheless, if the sperm maturational
events are not given time to occur prior to zona hardening, a reduced number
of 2-cell embryos or a complete fertilization block would be observed. The need
to incubate cryopreserved C57BL/6 sperm is in contrast to previous studies, in
which cooling and cryopreservation reduced the time necessary for sperm to ob-
tain fertilization competency[9], [18]. Conceivably, inherent genetic components
regulate the differences observed in this phenotype.
Figure 1.
Influence of sperm pre-incubation on in vitro fertilization success.
(a). Variability among males
was limited by creating 3 pools of sperm, each containing sperm from 2 C57BL/6J males. Sperm treatments
included cryopreservation in 3% skim milk, 18% raffinose (o); 3% skim milk, 18% raffinose supplemented
with 477 µM monothioglycerol (◊) and freshly collected in Cook's Mouse Vitro Fert (MVF;
). Sperm were
cryopreserved and thawed using previously optimized cooling and warming rates. Three IVFs per treatment,
each using cumulus oocyte masses from 2 superovulated C57BL/6J females (mean±standard deviation; 66±20
oocytes/IVF), were performed following 0, 30, 60, or 80 min of sperm incubation in MVF. Treatment effects
were determined using ANOVA and preplanned contrasts between consecutive time points. The arrow and
black boxed p-values indicate the reduced ability of cryopreserved C57BL/6J sperm to fertilize oocytes after 60
min of incubation when compared to freshly collected sperm using Dunnett's Method[32]. (b). Sperm were
collected from 6 C57BL/6J males and 3 pools created, each containing sperm from 2 males. The pools were
subdivided into 3 treatments: i) Control - no treatment (black); ii) Diluted live sperm - sperm were diluted 1:4
in MVF to mimic the concentration of live sperm equivalent to that found in cryopreserved samples (gray); and
iii) Diluted live+dead sperm - live sperm were diluted 1:4 with killed sperm (flash frozen in liquid nitrogen) to
simulate the number and ratio of live and dead sperm observed in the cryopreserved samples (white). The various
treatments received either no incubation or 60 min of incubation in MVF before adding cumulus oocyte masses
from two superovulated C57BL/6J oocytes (mean±standard deviation; 69±23 oocytes/IVF). In vitro fertilization
was done at least six times for each treatment, twice per pool of males. Comparisons indicated were evaluated
using preplanned contrast.
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