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Arkansas Animal Science Department Report 2001
+ T; Petr et al., 1996). After the initial 20 h of culture, COC's
in each treatment were rinsed and cultured to 46 h in M-199
medium supplemented with 0.1 mM glutathione, 10% FCS,
50 µg/ml gentamicin, and 0.05 NIH units of LH.
In both Experiments 1 and 2, approximately one-third
of the COC’s in each treatment were removed from culture
after the initial 20 h of culture, stripped of cumulus cells, and
then fixed and stained to assess stage of nuclear maturation.
At the termination of culture (46 h), the remaining COC's
were recovered and cumulus cells were mechanically
removed. Half of the resulting denuded oocytes were fixed
and stained to assess nuclear maturation, while the other half
were chemically activated and cultured to assess parthenogenetic
cleavage.
Oocytes in each treatment group were chemically activated
by exposure to NCSU-23 medium containing 50 µM
calcium ionophore A23187 for 3 min (Wang et al., 1999).
After exposure to activation treatment, the oocytes were
rinsed and placed into 4-well culture plates containing
NCSU-23 medium and cultured in a humidified atmosphere
of 5% CO2 in air at 39 ºC. Forty-eight hours after activation,
embryos were evaluated for cleavage and uncleaved oocytes
were discarded. On day 7, development to the morula and/or
blastocyst stage was assessed.
Experiment 1 and 2 were each replicated three times.
The JMP program, (SAS Institute Inc. Cary, NC) was used for
statistical analysis. Analysis of variance for a randomized
block design (blocked on replicate) was used with the
response variables being the percentage of GV, MII, cleaved
and morula/blastocysts, transformed by the angular transformation.
Pairwise comparisons of treatments were done by
multiple t-tests at the 5% level of probability.
Results and Discussion
The sequence of FSH and LH supplementation had no
effect on the percentage of oocytes remaining at the GV stage
at 20 h of culture (Table 1). Across treatments, the percentage
of oocytes maturing to MII at 46 h ranged from 38 to 61%.
Addition of both FSH and LH to culture medium after 20 h of
culture reduced maturation, when compared with other treatments.
Previous studies report that FSH supports estradiol
production by cumulus cells in the absence of significant levels
of LH and estradiol slows nuclear progression (Richter
and McGaughey, 1979). Luteinizing hormone promotes
luteinization of cumulus cells and a shift in steroid synthesis
from estradiol to progesterone. Progesterone in turn, promotes
nuclear progression (Eroglu, 1993). Therefore, we had
expected that the addition of FSH the first 20 h and LH the
second 26 h of maturation would slow or delay nuclear maturation
and allow both nuclear and cytoplasmic maturation to
be completed at approximately the same time. However, it
would appear that deletion of LH from maturation medium
the first 20 h of culture is not enough to delay GVBD.
Oocyte cleavage after activation ranged from approximately
29 to 50% (Table 2). As with maturation, addition of
both FSH and LH to culture medium after the initial 20 h of
culture reduced subsequent cleavage. These results tend to
confirm a previous study reporting that the presence of FSH
and LH during the last 20 h period of culture reduces the
oocytes’ ability to properly mature (Funahashi et al., 1994).
There were no differences among treatments for the percentage
of embryos developing into morulae and blastocysts.
However, adding FSH the first 20 h and LH the remaining 26
h numerically increased the number of blastocysts, when
compared with all other treatments. While this difference was
not statistically significant, it would likely be of economic
benefit in labs producing porcine embryos.
Based on the number of oocytes remaining at the GV
stage at 20 h of IVM, all of the chemical treatments more
effectively blocked GVBD than the control treatment (Table
3). The DMAP and dbcAMP + T treatments were equally
effective in blocking nuclear maturation. The DEX treatment
was less effective than DMAP but similar to dbcAMP + T in
blocking GVBD. Both DEX and dbcAMP + T treatments
appeared to be reversible and resulted in similar rates of maturation
to MII after removal from the maturation medium.
The DMAP treatment appeared to have an irreversible effect
on subsequent nuclear maturation. All treatments reduced
subsequent cleavage after parthenogenetic activation when
compared with the control treatment. The DMAP treatment
was most detrimental to subsequent maturation, cleavage and
development. The mean number of cells per developing
embryo were similar among treatments and within the range
reported for parthenogenetic embryos.
The objective of our studies was to block GVBD for a
period of 20 h, without detrimental effects to subsequent maturation
and cleavage. Blocking nuclear maturation at the germinal
vesicle stage has proven to be beneficial for development
to the blastocyst stage. A previous study reported that
the presence of dbcAMP during the first 20 h of culture for
maturation, induces a more synchronous meiotic progression
of porcine oocytes and improves the rate of early embryonic
development to the blastocyst stage after fertilization
(Funahashi et al., 1997). In our study, oocytes were parthenogenetically
activated rather than fertilized. Further study is
planned to determine if dbcAMP and testosterone treatment
can be used in oocyte maturation regimens to improve developmental
competence after fertilization.
Implications
It may be possible to improve porcine developmental
competence by synchronizing nuclear and cytoplasmic maturation
in vitro. The most effective method to synchronize
maturation is through use of chemical agents to delay GVBD.
Both DEX and dbcAMP + T treatments appeared to be
reversible and resulted in similar rates of maturation to MII
and cleavage. Additional studies are needed to assess developmental
competence of oocytes exposed to these treatments
after fertilization, rather than activation and parthenogenetic
cleavage.
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